Oligonucleotide-mediated quantitative multiplexed immunoassays

ABSTRACT

Methods and compositions for quantitative immunoassays are provided, in which ligand-conjugated probes are used to label samples and ligand-surfaced microspheres are used as quantitative reference standards. Certain embodiments provide a method of quantitative flow cytometry where ligands are oligonucleotides, and a sample comprising one or more cells is contacted with a hybridized antibody::fluorophore labeled targeting construct to label the cells, and the labeled cells are analyzed. In some embodiments, a population of quantitative labeled oligospheres labeled with the same fluorescent label as the cells is analyzed using the flow cytometer and used to create a quantitative standard curve of cytometer intensity versus molecules fluorescent label per oligosphere event. A standard curve trendline is established and used to determine the molecules of fluorescent label per cellular event for the antigen-positive cell populations. Based on molecules of fluorescent label per cell, the amount of Antibody Binding per Cell (ABC) is quantified.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/660,261, filed Jun. 15, 2012, herebyincorporated by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention relates generally to the fields of immunology,molecular biology, and cellular biology. More particularly, it relatesto quantitative multiplexed cellular analysis using flow cytometry,microscopy, and/or fluorimetry.

2. Description of Related Art

Multiplexed target labeling and analysis are principal strategiesapplied in molecular biology research. In general, surface orintracellular antigens indicative of cell status are detected bymultiplexed labeling with targeting reagents (e.g., antibodies),followed by visualization of the targeting reagents by specific labelingprobes (e.g., fluorophores). In some instances, targets in solution areanalyzed using a similar approach. The sample is analyzed by flowcytometry, microscopy, fluorimetry, or other instrumentation equipped tomeasure labeling probe signal.

Multiplexed targeting assays have been facilitated in the last severaldecades by an increasing variety of commercially available antibodiesbiochemically conjugated to labeling reagents. Fluorescent reagents arethe most common type of label used in the laboratory, although otherlabels may be utilized for specific applications (enzymes,radioisotopes, heavy metals, etc). Despite the growing availability ofdirectly-labeled targeting reagents, the majority of reagents are onlyavailable conjugated to a limited number of labels, often in the samestandard fluorophore such as FITC. This is particularly true of reagentstargeting novel or niche markers.

A variety of parameters must be considered in order to determine anoptimal multiplexed detection strategy, including cell type(s), targetdensities, labeling reagent characteristics, and instrumentspecifications. Limitations placed on label-target choice by commercialavailability, coupled with reliance on qualitative analysis parameters,can cause variation in results and subsequent interpretation of dataacross experiments, researchers, and laboratories.

The prevalence of qualitative, rather than quantitative, analysis inmany immunoassays is a result of several factors. Qualitative analysisis almost universally practiced with flow cytometric and microscopyassays, due to the nature of instrumentation, which are configured toprovide a measure of adjustable, relative intensity, rather than unitsof absolute intensity. While some quantitative technologies exist, suchas dyed fluorescent microspheres, at present these technologies requirean additional investment of cost and preparation time that may determany researchers, and even when utilized may not produce reliable andaccurate quantitative measures. Although immunoanalysis procedures are,by and large, executed by researchers with considerable experience andexpertise, there is no question that a streamlined method of accurate,quantitative analysis would represent a significant asset to thefield—notably, for flow cytometric applications which are particularlysubject to variation and error incurred by the qualitative approach.

As existing technologies are often time-consuming, cumbersome, andinaccurate, it is understandable that the quantitative analysis endeavoris not usually pursued by the research laboratory.

SUMMARY OF THE INVENTION

Various embodiments address challenges presented by conventionalqualitative immunoassay methods by utilizing DNA-directed assembly orother means by which complementary ligands pair to form a one-to-onecomplex for quantitative target labeling. In certain embodiments,antibodies are used as the targeting reagent, and oligonucleotides areused as the ligand. Antibody:oligonucleotide targeting constructs arehybridized to complementary oligonucleotide:label constructs to create alabeled targeting hybrid. The hybrid is then used to label antigens andprovide a signal for analysis. Alternatively, targeting constructs arefirst applied to a sample and then the labeling construct is applied,providing a signal for analysis. Labeled quantitative oligospheres areadded to the analysis and used to convert relative units of signalintensity provided by the label to absolute measures of Label Per Event(LPE). In certain embodiments, an event may comprise a single cell, avolume of solution, a concentration or volume of analyte, or a unit ofsurface area.

LPE is then used to quantify the number of targeting reagent moleculeswithin a sample based on known label-target ratio, which is establishedduring ligation of targeting construct to labeling construct. In certainembodiments in which the targeting reagent is an antibody, and thesample comprises a cellular preparation, the quantitative measure isnoted as Antibodies Bound per Cell (ABC).

As used herein, a ligand-surfaced microsphere refers to a microsphere towhich a ligand is conjugated. Non-limiting examples of ligands mayinclude oligonucleotides, peptides, or haptens. Specifically, an“oligosphere” refers to a microsphere to which oligonucleotides areconjugated for surface ligation.

Several techniques are known for conjugating ligands to microspheres.The ligand-microsphere conjugation procedure may involve modification ofamine, carboxyl, hydroxyl or other reactive groups on oligonucleotidesand microsphere surfaces in order to incorporate linker moieties forsubsequent conjugation reaction. Linker chemistry may include HyNic/4FB(hydrazone), (strept)avidin/biotin, phosphoramidite, octadinyl dU, andother chemistries. Alternatively, the microspheres may bepre-manufactured to present surface reactive groups to whichreactive-group bearing oligo may be conjugated (e.g., amino- orstreptavidin-modified microspheres). In certain aspects, a linkersequence is placed between the microsphere and the operative region ofthe oligonucleotide. Such linkers may, for example, facilitateconjugation to the microsphere and/or reduce steric hindrance of theoligonucleotide.

Microspheres are generally spherical particles with diameters in themicrometer range (i.e., 1 μm to 1,000 μm). For flow cytometerapplications, oligospheres with diameters between about 1-10 μm, 3-8 μm,or 3-6 μm, are preferred. Microspheres may be made from variousmaterials including, polymers (e.g., polyethylene or polystyrene),glass, or ceramic.

In certain aspects, the microspheres are magnetic. As used herein,“magnetic” includes paramagnetic and super paramagnetic. Themicrospheres may also be encoded. The size of the microspheres in asubpopulation may also be used to distinguish one subpopulation fromanother. Another method of encoding microspheres is to incorporate amagnetically responsive substance, such as Fe₃O₄, into the structure.Paramagnetic and superparamagnetic microspheres have negligiblemagnetism in the absence of a magnetic field, but application of amagnetic field induces alignment of the magnetic domains in themicrospheres, resulting in attraction of the microspheres to the fieldsource. Combining fluorescent dyes, microsphere size, and/ormagnetically responsive substances into the microspheres can furtherincrease the number of different subpopulations of ligand-conjugatedmicrospheres that can be created.

As used herein a “labeled oligosphere” refers to an oligosphere and alabeling construct, in which the respective oligonucleotides haveannealed to form a hybrid. As discussed, labeling constructs contain alabeling moiety and are designed to hybridize to the oligonucleotidesequences on the oligospheres. A number of techniques are known forattaching labeling moieties to nucleic acids. These techniques includethe use of a dextran scaffold bearing oligonucleotides and fluorophores,as well as the direct conjugation of the fluorophore conjugated to theoligonucleotide.

Compositions comprising a population of quantitative labeledoligospheres prepared according to the methods disclosed herein also areprovided.

As used herein a “targeting oligosphere” refers to an oligosphere and atargeting construct to which the respective oligonucleotides haveannealed to form a hybrid.

Certain embodiments provide a method of preparing a population ofquantitative labeled oligospheres comprising: (a) separately combiningat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more subpopulations ofoligospheres with at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or moredifferent concentrations of labeling constructs under conditionssuitable for the hybridization of the oligospheres to the probes toobtain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or moresubpopulations of labeled oligospheres; and (b) combining thesubpopulations of labeled oligospheres to obtain a titrated populationof quantitative oligospheres bearing known numbers of labeling moleculesat discrete and increasing saturations, providing a standard curveagainst which an unknown sample can be evaluated. The titratedpopulation of labeled oligospheres will, therefore, comprise at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more subpopulations of labeledoligospheres having different amounts of labeling moiety. In someembodiments, at most or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12or more subpopulations of oligospheres (or any range derivable therein)are combined with at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 or more different concentrations of labeling construct (or any rangederivable therein) under conditions suitable for the hybridization ofthe oligospheres to the labeling construct to obtain at least or at most2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more subpopulations of labeledoligospheres (or any range derivable therein). As used herein“quantitative oligospheres” means a population of labeled oligospherescontaining at least two different subpopulations of labeledoligospheres, as described herein.

Methods of preparing a population of quantitative oligospheres mayfurther comprise individually analyzing the subpopulations ofquantitative oligospheres by flow cytometry prior to combining thesubpopulations of quantitative oligospheres to obtain the titratedpopulation of quantitative oligospheres. This analysis may compriseanalyzing one or more parameters including, but not limited to, peakintensity, bandwidth, or peak separation of the subpopulations of thelabeled oligospheres. In embodiments where encoded microspheres areused, parameters relating to the encoding moieties (e.g., internalfluorescent dyes) may also be analyzed. In certain aspects relating toflow cytometry, the labeled oligospheres are gated on singlets and thenthe singlets are visualized as histograms. The histograms of thesubpopulations of labeled oligospheres may be overlayed.

Methods of preparing a population of labeled oligospheres may furthercomprise quantifying a Label-signal Per oligosphere Event (LPE) using amicroplate fluorimeter to measure sample intensity versus a standardcurve. In some embodiments, the label-signal is a fluorescent signal,and intensity is converted to LPE using a linear trendline equationprovided by a fluorescent standard curve. In certain aspects, methods ofpreparing a population of labeled oligospheres may further comprisedetermining the number of labeled oligospheres in a sample using ahandheld particle counter or other counting devices known to those inthe art.

The ligand-conjugated microspheres and antibody:ligand targetingconstructs disclosed herein may be used in numerous applicationsincluding, for example, Quantitative Flow Cytometry (QFC), spectralcompensation for polychromatic flow cytometry, reference standards forQuality Control (QC) of cytometric instrumentation (i.e. alignment orcalibration), single cell mass cytometry (CyTOF), microscopy, andEnzyme-Linked ImmunoSorbent Assays (ELISA). Microscopy applicationsinclude, for example, singleplex or multiplex QuantitativeImmunoCytoChemistry (Q-ICC) or ImmunoHistoChemistry (Q-IHC).

A variety of labeling moieties may be employed in the methods andcompositions disclosed herein. Non-limiting examples of labelingmoieties include biofluors (e.g., phycoerythrin (PE), allophycocyanin(APC), small molecule fluorophores (FITC, Alexa dyes, DyLight dyes,eFluor dyes, etc.), fluorescent proteins (GFP, CFP, YFP, mCherry, dsRed,etc.), or quantum dots. For CyTOF applications heavy metal or isotopelabeling moieties are preferred. For ELISA or ICC/IHC, enzymaticlabeling moieties may be used (e.g., horseradish peroxidase, alkalinephosphatase, etc), followed by a tertiary detection reagent (e.g.,fluorescent, colorimetric, or luminescent enzyme substrate). In someembodiments, radioisotopes may be used as a label.

Non-limiting examples of fluorophores include Alexa Fluor (e.g. AlexaFluor 488, 532, or 647), BODIPY® (e.g. BODIPY®-630/650, -650/665, -FL,R6G, -TMR, or -TRX) CyDye™ (e.g. Cy2™, Cy3™, or CyS™), DyLight™ (e.g.Dy490, Dy549, Dy649, and Dy405), acridine orange, coumarin, cyanine,fluorescein, resorufin, and rhodamine dyes. Other non-limiting examplesof fluorescent dyes include an orange fluorescent squarine dye such as2,4-Bis [3,5-dimethyl-2-pyrrolyl] cyclobutenediylium-1,3-diololate, ared fluorescent squarine dye such as 2,4-Bis[1,3,3-trimethyl-2-indolinylidenemethyl]cyclobutenediylium-1,3-dioxolate, or an infrared dye such as 2,4 Bis[3,3-dimethyl-2-(1H-benz[e]indolinylidenemethyl)]cyclobutenediylium-1,3-dioxolate. Further examples of fluorescent dyesinclude quantum dots, AMCA, Cascade Blue®, 6-FAM™, HEX™, 6-JOE, OregonGreen®, Pacific Blue™, REG, Rhodamine Green™, Rhodamine Red™, ROX™,TAMRA™, TET™, Tetramethylrhodamine (TMR), or Texas Red®. Fluorophoresmay include phycobilliproteins including, but not limited to,phycoerythrin (PE) and allophycocyanin (APC), or tandem-dye preparationsof phycobiliproteins (e.g. PE-Cy5 or APC-Cy7).

The sequences of the oligonucleotides used in the in the methods andcompositions disclosed herein are not limited to any particularsequence. Those of ordinary skill in the art will be able to determineappropriate sequences based on the assay conditions, particularlyhybridization conditions and the potential for undesirablecross-hybridization with other probes or sequences in the sample. It isgenerally desirable to use oligonucleotides that have low reactivitywith unmatched oligo sequences, high melting temperature, and stable androbust hybridization activity. It may also be desirable to useoligonucleotides that form hairpin structures. Preferably, oligos willnot hybridize to other nucleic acids in the sample during a reaction.The proper selection of non-cross hybridizing sequences is useful inassays, particularly assays in a highly parallel hybridizationenvironment, that require stringent non-cross hybridizing behavior. Incertain embodiments, the sequences are between 6 to 60, 8 to 50, 10 to40, 10 to 20, 12 to 24, or 20 to 30 nucleotides in length. Non-limitingexamples of such sequences include the sequences of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and theirrespective complementary sequences. The oligonucleotides may comprisenatural bases (A, T/U, G, and C) and/or non-natural bases (e.g., peptidenucleic acids (PNAs), locked nucleic acids (LNAs), iso-nucleotides).

Other embodiments provide an interchangeable labeling system comprising:(a) an antibody:oligonucleotide targeting construct comprising anantibody region and a first universal nucleic acid region; and (b) aplurality of different labeling constructs comprising a label and asecond universal nucleic acid region that is complementary to the firstuniversal nucleic acid region, wherein each of the plurality ofdifferent labeling constructs has a different label, but comprises thesame second universal nucleic acid region.

Further embodiments provide an antibody:oligonucleotide targetingconstruct comprising a first oligonucleotide, an oligosphere conjugatedto a second oligonucleotide comprising a sequence identical to thesequence of the first oligonucleotide, and a labeling constructcomprising a third sequence that is complementary to the first and thesecond oligonucleotides. In some embodiments, the first oligonucleotidecomprises a sequence selected from the group consisting of the sequenceof, or a sequence complementary to the sequence of, SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In someembodiments, the second oligonucleotide comprises a sequence selectedfrom the group consisting of the sequence of, or a sequencecomplementary to the sequence of, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, or SEQ ID NO: 10.

Other embodiments provide a composition comprising a titrated populationof labeled oligospheres, wherein the titrated population of labeledoligospheres comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12subpopulations of labeled oligospheres, wherein each of thesubpopulations of labeled oligospheres is hybridized to a differentamount of labeling construct.

Certain embodiments provide a method of quantitative flow cytometrycomprising: (a) contacting a sample comprising one or more cells with alabeled targeting hybrid under conditions suitable for binding of thelabeled targeting hybrid construct to an antigen on the cells; (b)analyzing the cells bound to a labeled targeting hybrid in the sampleusing a flow cytometer; (c) analyzing a population of quantitativelabeled oligospheres, wherein the population of quantitative labeledoligospheres is labeled with the same fluorescent label as the labeledtargeting hybrid construct; (d) determining a median, mean, or GeometricMean Fluorescent Intensity (GMFI) for each population of quantitativelabeled oligospheres; (e) creating a standard curve for quantitation oflabeled targeting hybrid by plotting GMFI vs known molecules of labelper microsphere event (LPE or FPE), LPE or FPE having been previouslyquantified fluorometrically; (f) determining the LPE or FPE for one ormore cell populations which bind the labeled targeting hybrid'stargeting moiety from the median, mean or GMFI of cellular event(s); (g)using the LPE or FPE to quantify the amount of labeled targeting hybridper cell (i.e., ABC).

In certain aspects, the population of quantitative labeled oligospheresand the cells bound to the labeled targeting hybrid are combined in thesample prior to analyzing the mixed population of quantitative labeledoligospheres and cells bound to the labeled targeting hybrid in the flowcytometer. In some aspects, the population of quantitative labeledoligospheres are analyzed in the flow cytometer before or after thecells bound to the labeled targeting hybrid are analyzed in the flowcytometer. In other aspects, cells bound to an unhybridized targetingconstruct and unhybridized oligospheres bearing increasing titrations offree oligonucleotide are combined in the presence of an excess oflabeling construct comprising a complementary oligonucleotide to thetargeting construct and hybridization is allowed to proceed, followed byflow cytometric analysis of the mixed sample of cells bound to newlabeled targeting hybrid and oligospheres.

In one embodiment, a method of quantitative flow cytometry is providedcomprising: (a) contacting a sample comprising one or more cells with alabeled targeting hybrid under conditions suitable for binding of thelabeled targeting hybrid construct to an antigen on the cells; (b)contacting the sample with a population of quantitative oligosphereswherein the population of quantitative oligospheres is labeled with thesame labeling moiety as the labeled targeting hybrid; (c) analyzing apopulation of quantitative labeled oligospheres and the cells bound tothe labeled targeting hybrid in the sample in a cytometer; (d)determining a median, mean, or Geometric Mean Fluorescent Intensity(GMFI) for each population of quantitative labeled oligospheres; (e)creating a standard curve for quantitation of labeled targeting hybridby plotting GMFI vs known molecules of label per microsphere event (LPEor FPE), the LPE or FPE having been previously quantifiedfluorometrically; (f) determining the LPE or FPE for one or more cellpopulations which bind the labeled targeting hybrid's targeting moietyfrom the median, mean or GMFI of cellular event(s); and (g) using theLPE or FPE to quantify the amount of labeled targeting hybrid per cell(i.e., ABC).

In another embodiment, a method of quantitative flow cytometry isprovided comprising: (a) contacting a sample comprising one or morecells with an unlabeled targeting construct under conditions suitablefor binding of the targeting construct to an antigen on the cells; (b)contacting the sample with a population of unlabeled oligospheres; (c)contacting the mixed sample of cells and oligospheres with sufficientlabeling construct to hybridize to oligospheres and targetingconstructs, thereby creating quantitative oligospheres labeled with thesame labeling moiety as the targeting construct bound to antigen on thecells; (d) analyzing a population of quantitative labeled oligospheresand labeled cells in the sample in a cytometer; (e) determining amedian, mean, or Geometric Mean Fluorescent Intensity (GMFI) for eachpopulation of quantitative labeled oligospheres; (f) creating a standardcurve for quantitation of labeled targeting hybrid by plotting GMFI vsknown molecules of label per microsphere event (LPE or FPE), LPE or FPEhaving been previously quantified fluorometrically; (g) determining theLPE or FPE for one or more cell populations which bind the labeledtargeting hybrid's targeting moiety from the median, mean or GMFI ofcellular event(s); (g) using the LPE or FPE to quantify the amount oflabeled targeting hybrid per cell (i.e., ABC).

Another embodiment provides a method of flow cytometric spectralcompensation comprising: (a) analyzing at least two populations ofquantitative labeled oligospheres in the flow cytometer bearing a singlelabel in each population; (b) obtaining cytometric data in at least twocytometric detector channels for all labeled oligospheres beinganalyzed; (c) utilizing cytometric data acquisition and/or analysissoftware to determine spectral compensation parameters using labeledoligosphere data; and (d) applying compensation parameters to cellslabeled with at least two label-target hybrids bearing the same labelsas the labeled oligospheres used to determine compensation parameters.

Other embodiments provide a method of calibration of cytometricinstrumentation comprising: (a) analyzing at least one population ofquantitative labeled oligospheres in a flow cytometer; (b) obtainingcytometric data in at least one cytometric detector channel; (c)utilizing known degree-of-labeling data of quantitative oligospheres toevaluate sensitivity and resolution of the instrument; and (d)performing calibration and alignment procedures based on observedsignaling of labeled oligospheres.

A further embodiment provides a method of quantitativeimmunocytochemistry comprising: (a) contacting a sample comprising oneor more cells with a labeled targeting hybrid under conditions suitablefor binding of the labeled targeting hybrid to a cellular target; (b)contacting the labeled cell sample with a population of quantitativelabeled oligospheres bearing the same label as the labeled targetinghybrids applied to the cells; (c) analyzing the sample using amicroscope equipped with an appropriate fluorescent filter to observethe fluorescent signal of the labeled cells and microspheres, using acamera and imaging software to obtain representative images of thesample; (d) utilizing image-analysis software to create asignal-to-noise threshold and intensity standard curve using fluorescentoligospheres; (e) utilizing the signal intensity data provided by thelabeled oligospheres to quantitate signal intensity of labeled cells;and (f) converting cell signal intensity units to hybrid-per-cell unitsby (signal intensity/label-target DOL).

One embodiment provides a method of quantitative immunocytochemistrycomprising: (a) contacting a sample comprising one or more cells with atleast a first and a second labeled targeting hybrid under conditionssuitable for binding of the hybrid to a cellular target; (b) contactingthe labeled cell sample with a population of at least a first and asecond population of quantitative labeled oligospheres bearing the samelabel as the labeled targeting hybrids applied to the cells; (c)analyzing the sample using a microscope equipped with an appropriatefluorescent filter to observe the fluorescent signal of the labeledcells and microspheres, using a camera and imaging software to obtainrepresentative images of the sample; (d) utilizing image-analysissoftware to create a signal-to-noise threshold and intensity standardcurve using fluorescent oligospheres; (e) utilizing the signal intensitydata provided by the labeled oligospheres to quantitate signal intensityof labeled cells; and (f) converting cell signal intensity units tohybrid-per-cell units for each label-target hybrid applied by (signalintensity/label-target DOL).

Another embodiment provides a method of quantitative immunocytochemistrycomprising: (a) contacting a sample comprising a tissue sample with alabeled targeting hybrid under conditions suitable for binding of thehybrid to a target on or within the tissue; (b) contacting the labeledtissue sample with a population of quantitative labeled oligospheresbearing the same label as the labeled targeting hybrids applied to thetissue; (c) analyzing the sample using a microscope equipped with anappropriate fluorescent filter to observe the fluorescent signal of thelabeled tissue and microspheres, using a camera and imaging software toobtain representative images of the sample; (d) utilizing image-analysissoftware to create a signal-to-noise threshold and intensity standardcurve using fluorescent oligospheres; (c) utilizing the signal intensitydata provided by the labeled oligospheres to quantitate signal intensityof labeled tissue; (f) converting tissue signal intensity units tohybrid-per-area units by (signal intensity/label-target DOL).

In one embodiment, there is provided a method of quantitativeimmunohistochemistry comprising: (a) contacting a sample comprising atissue sample with at least a first and a second labeled targetinghybrid under conditions suitable for binding of the hybrid to a targeton or within the tissue; (b) contacting the labeled tissue sample with apopulation of at least a first and a second population of quantitativelabeled oligospheres bearing the same label as the labeled targetinghybrids applied to the tissue; (c) analyzing the sample using amicroscope equipped with an appropriate fluorescent filter to observethe fluorescent signal of the labeled cells and microspheres, using acamera and imaging software to obtain representative images of thesample; (d) utilizing image-analysis software to create asignal-to-noise threshold and intensity standard curve using fluorescentoligospheres; (c) utilizing the signal intensity data provided by thelabeled oligospheres to quantitate signal intensity of labeled tissue;(f) converting tissue signal intensity units to hybrid-per-area unitsfor each label-target hybrid applied by (signal intensity/label-targetLabel Per Event (LPE)). The microscope may be, for example, aconventional inverted fluorescent microscope, a high-content scanningmicroscope, or a cytometric microscope.

Other embodiments provide methods of quantitative Enzyme-LinkedImmunoSorbent Assay (ELISA) comprising: (a) contacting a sample with alabeled targeting hybrid in a microplate under conditions suitable forbinding of the hybrid to a target presented by the sample; (b)introducing quantitative labeled oligospheres to the microplate; (c)analyzing the microplate using a fluorimeter, luminometer, orspectrophotometer to determine labeling intensity of the sample and theoligospheres; and (d) utilizing the signal intensity data provided bythe labeled oligospheres to convert sample labeling intensity to knownnumber of targets per cell based on oligosphere Label Per Event (LPE).The method may further comprise applying a detection reagent to thesamples to visualize the label. The detection reagent may be, forexample, a fluorescent, luminescent, or colorimetric enzymaticsubstrate.

In other aspects, the targeting agent may be attached to themicrosphere. For example, one embodiment provides a method ofquantitative microsphere-based targeting assay comprising: (a)contacting a population of unlabeled oligospheres with increasingtitrations of a labeling construct; (b) introducing quantitativetargeting oligospheres to a sample under conditions suitable for bindingof the labeling construct to a target on or within the sample; (c)applying a detection reagent to all samples to visualize the binding ofthe target to the oligospheres; (d) analyzing the oligospheres using acytometer or particle analyzer; (e) analyzing a population ofquantitative oligospheres using the cytometer or particle analyzer; and(e) utilizing the signal intensity data provided by the quantitativeoligospheres to convert intensity of labeled targeting oligospheres toknown number of targets per sphere. The detection reagent may comprise,for example, a fluorescent antibody reactive with the target, or a firstantibody reactive with the target and a fluorescent second antibodyreactive with the first antibody.

The method may be multiplexed by using additional (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 15, 20, 25 or more) labeled targeting hybrids andlabeled oligospheres. For example, the method of quantitative flowcytometry may comprise: (a) contacting the sample with at least a firstand a second labeled targeting hybrid, wherein the first labeledtargeting hybrid comprises an antibody and a fluorescent label thatdiffer from the antibody and the fluorescent label of the second labeledtargeting hybrid, under conditions suitable for binding of the first andthe second labeled targeting hybrid to their respective binding sites onthe cells; (b) analyzing the cells bound to labeled targeting hybrid inthe sample in the flow cytometer; (c) analyzing at least a first and asecond population of quantitative oligospheres, wherein the fluorescentlabels of the first and the second populations of quantitativeoligospheres differ from each other, but are the same as the fluorescentlabel of either the first or the second labeled targeting hybrid, in aflow cytometer; (d) determining the Geometric Mean Fluorescent Intensity(GMFI) versus LPE trendline from the GMFIs of at least two differentpopulations of quantitative oligospheres; (e) determining the LPE forthe one or more cell populations bound to either the first or the secondlabeled targeting hybrid from the GMFI versus LPE trendlines; and (f)quantifying the amount of the first or the second labeled targetinghybrid bound per cell. In some embodiments, the first or the secondlabeled targeting hybrid comprise an antibody:oligonucleotide targetingconstruct, and bind an antigen on the cells.

Any of the compositions disclosed herein may be provided in a kit. Incertain embodiments, the kit comprises a composition comprising atitrated population of labeled oligospheres, wherein the titratedpopulation of labeled oligospheres comprises at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 subpopulations of labeled oligospheres, wherein eachof the subpopulations of labeled oligospheres is hybridized to adifferent amount of labeling construct. In certain aspects, the titratedpopulation of labeled oligospheres are combined in a single container inthe kit. In other aspects, the subpopulations are provided of labeledoligospheres are provided in separate containers in the kit. In someembodiments, the kit comprises an antibody:oligonucleotide targetingconstruct and/or a labeling construct.

The sample may be any sample that is suspected of containing an analyteof interest. In certain aspects the sample may be obtained from asubject who is being screened for the presence or absence of an antigenof interest. In another aspect, the sample may be from a subject who isbeing tested for the presence or absence of a pathogen. Where the sampleis obtained from a subject, it may be obtained by methods known to thosein the art such as aspiration, biopsy, swabbing, venipuncture, spinaltap, fecal sample, or urine sample. In some aspects of the invention,the sample is an environmental sample such as a water, soil, or airsample. In other aspects of the invention, the sample is from a plant,bacteria, virus, fungi, protozoan, or metazoan. In certain embodiments,the sample is a blood sample. The blood sample may be a whole bloodsample or it may be separated into various blood components. In certainembodiments, the sample is from the huffy coat.

The samples may contain cells that express antigens recognized by one ormore antibody:ligand targeting constructs. In certain embodiments, thecells are immune cells. The immune cells may be myeloid cells, such asmonocytes, macrophages, and dendritic cells (DC), or lymphoid cells,such as T cells, NK cells, B cells, and lymphoid DC. In otherembodiments the cells are cancer cells.

The antibody in the antibody:ligand targeting construct may comprise anantibody that specifically binds to any antigen of interest. In certainembodiments, the antigen of interest is an antigen that ischaracteristic of immune cells or cancer cells. Non-limiting examples ofantigens characteristic of immune cells are CD4, CD8, CD28, CD43, CD56,and CD62L. In particular embodiments, combinations of antibody:ligandtargeting constructs are employed. For example, in one aspect a firstantibody:oligonucleotide targeting construct comprises an antibody thatbinds to CD4 and the second antibody:oligo targeting construct comprisesan antibody that binds to CD8. Additional antibody:ligand targetingconstructs may be employed, such as at least a third and a fourthdifferent antibody:ligand targeting construct under conditions suitablefor binding of the third and the fourth antibody:ligand targetingconstructs to their respective antigens on the cells. Thus, for example,the first antibody:oligonucleotide targeting construct comprises anantibody that binds to CD4, the second antibody:oligonucleotidetargeting construct comprises an antibody that binds to CD8, the thirdantibody:oligonucleotide targeting construct comprises an antibody thatbinds to CD43, and the fourth antibody:oligonucleotide targetingconstruct comprises an antibody that binds to CD62L.

As used herein, the term “bioconjugate” means a construct in which atleast one biomolecule is attached to another moiety. In certainembodiments, bioconjugates may be proteins attached to ligands,including oligonucleotides. In other embodiments, bioconjugates may beligands attached to a labeling moiety. Attachment may occur by any ofthe linker chemistries discussed herein. Bioconjugates include, forexample, targeting constructs and labeling constructs.

As used herein, the term “targeting construct” means a construct inwhich a targeting moiety is attached to a ligand. In certainembodiments, the targeting construct is an antibody attached to anoligonucleotide. In other embodiments, the targeting construct is anon-antibody protein with the desired affinity for a particular bindingtarget attached to an oligonucleotide. As used herein, an “[X]:[Y]targeting construct” refers to a targeting construct in which atargeting moiety of type [X] is attached to a ligand of type [Y].

As used herein, the term “labeling construct” means a construct in whicha labeling moiety is attached to a ligand. In certain embodiments, thelabeling construct is a small molecule fluorophore attached to anoligonucleotide, optionally via a dextran or other scaffold. In otherembodiments, the labeling construct is a radionucleotide attached to anoligonucleotide, optionally via a dextran or other scaffold. As usedherein, an “[U]:[V] labeling construct” refers to a labeling constructin which a labeling moiety of type [U] is attached to an oligonucleotideof type [V]. Where [V] is stated as oligonucleotide, any sequence ofoligonucleotide is contemplated.

As used herein, the term “labeled targeting hybrid” means a targetingconstruct and a labeling construct, in which the ligands areoligonucleotides, and in which the respective oligonucleotides haveannealed to form a hybrid. In certain embodiments, this is anantibody:oligonucleotide targeting construct hybridized to acomplementary oligo:fluorophore labeling construct. As used herein, an“[M]::[N] labeled targeting hybrid” refers to a labeled targeting hybridin which a targeting construct containing a targeting moiety of type [M]is hybridized to a labeling construct containing a labeling moiety oftype [N]. As used herein, the term “antibody” is intended to referbroadly to any immunologic binding agent, such as IgY, IgG, IgM, IgA,IgD and IgE, and includes monoclonal antibodies, polyclonal antibodies,antibody fragments (Fab′, Fab, F(ab)₂, single domain antibodies (DABs),Fv, scFv (single chain Fv), and the like, and chimeric antibodies.

Any of the methods disclosed herein may be automated in whole or inpart. In some embodiments, computer executable instructions or acomputer readable medium comprising computer executable instructions,are provided for carrying out the steps of the methods disclosed herein.In certain aspects, the computer executable instructions comprise all orpart of one or more of the algorithms in FIGS. 11A-11B.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, amethod, composition, kit, or system that “comprises,” “has,” “contains,”or “includes” one or more recited steps or elements possesses thoserecited steps or elements, but is not limited to possessing only thosesteps or elements; it may possess (i.e., cover) elements or steps thatare not recited. Likewise, an element of a method, composition, kit, orsystem that “comprises,” “has,” “contains,” or “includes” one or morerecited features possesses those features, but is not limited topossessing only those features; it may possess features that are notrecited.

Any embodiment of any of the present methods, composition, kit, andsystems may consist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1C. (FIG. 1A) shows the antibody-oligonucleotide conjugation byHyNic-4FB chemistry: (i) Succinimidyl-6-hydrazinonicotinamide acetonehydrazone (S-HyNic) is added to purified antibody (Ig), allowingsuccinimydyl groups to react with free amino sites on lysine groups atthe antibody hinge region to form Ig-HyNic (iii). Similarly,succinimidyl-4-formylbenzamide (S-4FB) reacts with amino-modifiedoligonucleotide (ii) resulting in 4FB-oligo (iv). The HyNic- and4FB-modified biomolecules are then combined in the presence of aniline,which catalyzes the HyNic-4FB reaction, resulting in formation of acovalent hydrazone bond (v) and a stable antibody-oligonucleotidebioconjugate. (FIG. 1B) shows a scheme for the preparation of anoligo:fluorophore labeling construct. To prepare a 1:1 oligo:dextranconjugate (i), a 70 kDa amino-dextran bearing approximately 20 aminogroups per dextran is first reacted with an amount of S-HyNic sufficientto create 3-4 HyNic moieties per dextran, leaving >10 amino groupsavailable for downstream NHS-fluorophore labeling. In order to limit thefinal average oligo-per-dextran to <1, a stoichiometrically-limitingamount of 4FB-modified oligonucleotide is then added to theHyNic-dextran, in a pH 5.0 buffer containing 10% aniline catalyst (v/v).Following the 4FB/HyNic reaction, the oligo-conjugated amino-dextran ispurified first by size exclusion chromatography (SEC) to remove excessoligo, and then by ion exchange column (IEC) to remove unconjugateddextran. To the amino-dextran-oligo is then added a molar excess ofNHS-ester fluorophore (ii). Excess fluorophore is removed by dialysis,and the final oligo-dextran-fluorophore product is characterized by A260assay to confirm oligo-dextran ratio and fluorophore degree of labeling(DOL). (FIG. 1C) illustrates multiplexed cell labeling using labeledtargeting hybrids. Antibody:oligonucleotide targeting constructs arebriefly hybridized in solution to complementaryfluorophore:oligonucleotide labeling constructs (i) to form individualantibody::fluorophore labeled targeting hybrids (ii). The labeledtargeting hybrids may then be used to label cells for a single antigen,or as shown here, combined and used for multiplexed cell labeling (iii).

FIGS. 2A-2E Optimization of hybridization labeling conditions. (FIG. 2A)To determine optimal oligo/oligo ratio for hybridization of labeledtargeting hybrids, a titration of oligo:fluorophore labeling constructwas performed by adding 0.5-10 molar equivalents of oligo-1′:Dy490labeling construct to a fixed amount (6 pmol) of antibody:oligotargeting construct, αCD4:oligo-1, in a small volume of PhosphateBuffered Saline (PBS). The labeled targeting hybrids were then added toviable splenocytes to label CD4 antigen, and the labeled cells wereanalyzed by flow cytometry. Results showed the population of CD4+ cellsto be similar for all titrations; however, nonspecific background causedby addition of excess fluorophore increased above 1.0 oligo/oligoequivalents. A titration of 0.5 molar equivalents oligo:fluorophorelabeling construct was used for subsequent assays. (FIG. 2B)Hybridization was conducted either in solution, by combiningantibody:oligo and oligo:fluor in a small volume of PBS and then usingthe construct to label cells, or in situ by first cell-labeling withantibody:oligo and then introducing oligo:fluors for hybridization.Results showed very similar positive labeling percentages forhybridization in solution (dark green histogram) vs in situ (light greentinted histogram). Unstained cells are shown as a background control.(FIG. 2C) Blocking hybridization using an unmatched oligo sequence (bluehistogram) was successful, an indication that antigen labeling is highlyspecific using hybridized labeling constructs (green histogram).Blocking was conducted using a 5-fold molar excess of oligo:fluor asshown in panel (A). The blocking oligo did not prevent nonspecificbinding of the oligo:fluor, as evidenced by similar levels of dyebackground. (FIG. 2D) Time and temperature conditions for hybridizationwere investigated, using 15-60 minute hybridization at 4° C. (bluehistograms), 24° C. (gray histograms), or 37° C. (pink histograms).Results indicate that hybridization occurs with little variation overthis range of time and temperature conditions. (FIG. 2E) Adjustingsignal intensity by increasing fluorophore degree of labeling (DOL) from3-15 fluors per oligo:fluorophore labeling construct shows optimalsignal to background at DOL ˜7, with decreasing positive peak resolutionat DOL <7 and marked decreased in median fluorescence intensity (GMFI)at DOL >10, most likely due to fluorescence self-quenching.

FIGS. 3A-3B (FIG. 3A) Antigen detection by antibody::fluorophore labeledtargeting hybrids. Labeled targeting hybrids (i) xCD4::Dy490, (ii)xCD8::Dy549, (iii) xCD43::Dy649, and (iv) xCD62L::Dy405 wereprehybridized, mixed, and used to label cells (tinted histograms).Single-construct stains (untinted histograms), oligo:fluorophore-onlystains (gray histograms), and unstained cells (black histograms) werealso analyzed as controls. Percentages shown are for the 4-plex stainedcell sample. Results show effective antigen staining, comparable insingle-stained samples to multiplexed stained cells. Antigen-positivepopulation values were within expected ranges. (FIG. 3B) Multiplexedcell labeling data. 2-color dot plots depict multi-antigen labeling datafor cells stained with four antibody::fluorophore labeled targetinghybrids as previously described. The staining distributions seen hereprovide evidence that the system is specific and sensitive, allowing foraccurate gating and analysis of immune cell phenotypes. (I) CD4+ andCD8+ T-cell populations within the gated lymphocyte population wereclearly defined. (ii) The majority (˜74%) of lymphocytes are CD43+, andnearly all CD4+ cells were CD43+. Two CD43high populations were evident,either CD4− (34%) or CD4+ (7%). (iii) Most lymphocytes were CD62L+(˜75%). (iv) Gating of CD4+ lymphocytes and display of CD4+/CD43 vsCD4+/CD62L distribution reveals that 30% of CD4+ T-lymphocytes wereCD4+/CD43+/CD62L−, while 64% were triple-positive for all 3 antigens.Only a small minority of CD4+ cells were negative for CD43 (6%) or weredouble-negative for CD43 and CD62L (2%). (v) A defined population ofCD8+/CD43+ cells was visible, as well as a CD8− population of CD43+lymphocytes, either CD43low (29%) or CD43high (15%). (vi) A distinctpopulation of CD8+/CD62L+ cells are visible (26%). (vii) Most CD43+lymphocytes are CD62L+; a distinct population of CD43high CD62L+ cellswas evident (33%). (viii) Gating of CD8+ lymphocytes and display ofCD4+/CD43 vs CD4+/CD62L distribution reveals that the majority (93%) ofCD8+ lymphocytes were CD43+/CD62L+.

FIGS. 4A-4C. Interchangeable fluorophore hybridization using theuniversal oligo sequence pair. (FIG. 4A) Schematic showinginterchangeable hybridization principle. Antibody:oligo targetingconstruct (Ig:oligo-A) can be hybridized to any oligo-A′:fluorophorelabeling construct, resulting in antibody::fluorophore labeled targetinghybrid in a variety of spectra. (FIG. 4B) Universal-oligo constructswere used to label cells for control antigen CD4 in four distinctspectra. Results show that labeling percentages were very similar acrossfluorescent channels for both antigens, indicating that antibodies canbe effectively labeled in a variety of spectra using the universal-oligoapproach. (FIG. 4C) CD4:oligonucleotide and CD8:oligonucleotidetargeting constructs were combined for double-staining of cells in twofluorophore combinations: (i) xCD4::Dy490+xCD8::Dy649; (ii)xCD4::Dy405+xCD8::Dy549. In order to block oligo-mediated exchange ofoligo:fluorophores when constructs were mixed, an excess of unmodifiedoligo-A was added to each construct immediately following hybridizationin solution. While exchange was observed to be low (˜1%) withoutblocking oligo at typical staining conditions (data not shown), with theaddition of blocking oligo the exchange dropped to a negligible ˜0.5%.

FIG. 5A-5D. Preparation and analysis of quantitative oligospheres. (FIG.5A) Method I, parallel labeling of quantitative oligospheres alongsidecells. First, oligonucleotide-saturated microspheres (μ) are hybridizedto discrete, known amounts of complementary oligo:fluorophore labelingconstruct at increasing titrations (1-4). Amount of oligo-fluorophorelabel per microsphere event (LPE) is separately confirmed byfluorimetry. The labeled oligospheres are then added to cells which havebeen labeled with antibody-fluorophore targeting hybrids. The labeledoligospheres and cells are then cytometrically analyzed. (FIG. 5B)Method II, combined labeling of quantitative oligospheres in solutionwith cells. First, oligonucleotides are conjugated to microspheres (μ)at increasing, known surface saturations (1-4). The oligospheres areadded to cells which have been incubated with antibody-oligo targetingconstructs. The combined oligospheres and cells are then labeled insolution followed by cytometric analysis. (FIG. 5C) Fluorometricanalysis of four oligosphere populations hybridized with increasingtitrations of labeling construct (1-4, labeled low-high) as in FIG. 5A.Labeling construct Per oligosphere Event (LPE) is determined bymeasuring oligosphere fluorescence for a sample of oligospheres vs astandard curve of labeling construct in solution (not shown). Theoligospheres are then counted (not shown). LPE=[(mol labeling constructper sample×6E23 molecules per mol)/number oligospheres per sample].(FIG. 5D) Cytometric analysis of four oligospheres populations shown inFIG. 5C, 1-4, labeled low-high (solid filled histograms). Increasing LPEtranslates to increasing fluorescence when cytometer fluorescence dataare visualized by analysis software. Oligosphere singlets were gated(not shown) and data histograms were overlaid with a histogram showingunlabeled microsphere signal (autofluorescence, dashed open histogram).

FIGS. 6A-6D. Multiplexed Quantitative Flow Cytometry. Viable murinesplenocytes (filled histograms) were probed using four distinct labelinghybrids: anti-CD4::Dy490 (FIG. 6A), anti-CD8::Dy549 (FIG. 6B),anti-CD43::Dy649 (FIG. 6C), and anti-CD62L::Dy405 (FIG. 6D).Quantitative fluorophore-hybridized oligospheres (open histograms) werelabeled and analyzed with cells to quantify multiple surface antigens.

FIGS. 7A-7D. ABC Calculations. Geometric Mean Fluorescence Intensities(GMFIs) were determined for quantitative oligospheres (circles) in eachfluorescent channel using cytometric data analysis software. Log 10GMFIs were plotted vs log known fluorescent Label Per oligosphere Event(LPE), which were previously determined fluorimetrically (not shown).Cellular populations of interest (stars) were gated and GMFIs weredetermined using cytometric data analysis software. Antibody Binding perCell (ABC) A 1:1 label:antibody ratio is assumed in this system;therefore, LPE=ABC, and thus ABC can then be determined from GMFI usingthe equations shown. Cellular data points shown represent geometric meanABC for populations of interest (CD4+, CD8+, CD43^(LO), CD43^(HI),CD62L+).

FIG. 8. Single-cell ABC. Determination of single-cell ABC for 1,000lymphocytes was conducted as shown in FIG. 7 and results are presentedin 2-channel dot plots showing distribution of cellular populations. Asexpected, quantitative ABC cellular distribution is similar toqualitative 2D plots shown in FIG. 3B, yet quantitative data yieldsimproved information regarding the antigenicity of cells.

FIGS. 9A-9C. Oligospheres and Cells Labeled in Combination. Quantitativeoligospheres (open histograms) and cells (filled histograms) werelabeled in combination as shown in FIG. 5B. Results show that labelingin combination is feasible and produces distinctly labeled cellular andoligosphere populations. (FIG. 9A) Distinct populations of oligospheresand lymphocytes shown in a FSC vs SSC scatter plot. (FIG. 9B)Fluorescent (Alexa Fluor 488) lymphocytes displaying CD4− and CD4+populations. (FIG. 9C) Histogram overlay of oligosphere (black) andcellular (gray) data.

FIGS. 10A-10B. Quantitation of ABC_(CD4) Using Commercial Reagents.Commercial quantitative fluorescent microspheres (BD QuantiBrite PE)were used to quantify ABC_(CD4) using similar methodology and the samemonoclonal antibody (GK1.5) used for ABC_(CD4) quantitation using novelquantitative oligospheres. (FIG. 10A) Commercial microspheres andanti-CD4:PE stained cells were cytometrically analyzed to obtain GMFIdata. (FIG. 10B) Microsphere Log 10 GMFI plotted vs Log 10 PE moleculesper microsphere (lot-specific data provided by manufacturer). Theequation generated by the microsphere standard curve was then used toquantify mean CD4+ according to the manufacturer protocol. ABC_(CD4)data were very similar for commercial vs novel method (29741 vs 28824CD4 antibody per cell).

FIGS. 11A-11B. Flowcharts for Software Algorithms. (FIG. 11A) Flowchartfor Algorithm I (calculation of standard curve from oligosphere data).(FIG. 11B) Flowchart for Algorithm II (calculation of Antibody Bindingper Cell, ABC).

FIGS. 12A-12C. Spectral Compensation. Fluorophore-hybridizedoligospheres were used to spectrally separate two adjacent fluorescentchannels (FL1, FL2) using a conventional cytometer (BD LSRII) andcommonly used analysis software (FlowJo). (FIG. 12A) Oligospheres arerecognized by FlowJo Compensation Wizard software function, whichauto-gated the oligospheres for singlets, FL1/FL2 positive and FL1/FL2negative populations according to common methodology. The CompensationWizard created a compensation correction matrix (not shown) which wasthen applied to correct the mixed two-color oligosphere sample shownbelow. (FIG. 12B) Uncompensated mixed sample of FL1+ or FL2+oligospheres. Uncompensated data indicate 2 populations of FL1+ FL2+oligospheres rather than separate, single-fluorophore spheres. (FIG.12C). Compensated mixed sample of FL1+ or FL2+ oligospheres. Thecompensated data correctly show two separate, single-fluorophoreoligosphere populations (either FL1+ or FL2+, not FL1+FL2+).

FIGS. 13A-13B. Cytometer Alignment. Fluorophore-hybridized oligospheresof a single color and intensity (FIG. 13A) were compared to commercialfluorescent microspheres (FIG. 13B) in terms of CV (%) to evaluatewhether oligospheres may be used for instrument alignment. CVs weresimilar for oligospheres and commercialized microspheres. The inventorsplan to reduce CVs for oligospheres in the future by utilizingalternative amino-functionalized microspheres as a starting point, thatmay enable CV reduction of resulting fluorescent signal.

FIGS. 14A-14B. Cytometer Calibration. Fluorophore-hybridizedoligospheres of a single color and multiple intensities (FIG. 14A) werecompared to commercial fluorescent microspheres (FIG. 14B) in terms offluorescent peak resolution and distribution to evaluate whetheroligospheres may be used for instrument calibration. Oligosphere peakswere well distributed, but resolution was somewhat lower thancommercialized microspheres. As noted in FIG. 13, the inventors plan toimprove resolution for oligospheres in the future by utilizingalternative amino-functionalized microspheres as a starting point, thatmay improve resolution of resulting fluorescent signals.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Methods and composition for quantitative flow cytometry and quantitativeCyTOF are provided herein. Particular embodiments utilize a DNA-directedassembly strategy for cellular labeling. In certain aspects,antibody:oligonucleotide targeting constructs are hybridized tocomplementary oligonucleotide:fluorophores labeling constructs insolution to create a labeled targeting hybrid. The antibody::fluorophorelabeled targeting hybrid is then used to label cellular antigens.Fluorophore-hybridized oligospheres utilizing the same fluorophore usedto label the hybridized antibody::fluorophore labeled targeting hybridare added to the cytometric analysis in order to convert relative unitsof fluorescence to quantitative measures of Label Per Event (LPE). LPEis then used to calculate the number of Antibodies Bound per Cell (ABC)based on the known label-target ratio established during the constructligation step.

A. Flow Cytometry

Various embodiments described herein provide a quantitative approach toflow cytometry. Flow cytometry is an optical technique that analyzesparticles in a fluid mixture based on the particles' opticalcharacteristics using an instrument known as a flow cytometer. Flowcytometers hydrodynamically focus a fluid suspension of particles into athin stream so that the particles flow down the stream in substantiallysingle file and pass through an examination zone. A focused light beam,such as a laser beam illuminates the particles as they flow through theexamination zone. Optical detectors within the flow cytometer measurecertain characteristics of the light as it interacts with the particles.Commonly used flow cytometers can measure forward light scatter(generally correlated with the refractive index and size of the particlebeing illuminated), side light scatter (generally correlated with theparticle's internal complexity and granularity), and particlefluorescence at one or more wavelengths.

The types of “particles” that may be analyzed by a flow cytometerinclude cells as well as man-made microspheres or beads. Fluorescentmicrospheres for use as calibrants for semi-quantitative flow cytometryare generally known in the art and may be obtained from manufacturerssuch as Becton Dickinson (BD), Spherotech, and Bangs Laboratories.Protein-binding microspheres may also be analyzed via flow cytometry andare available from manufacturers such as Life Technologies (Invitrogen)and EMD-Millipore (Luminex).

Conventional methods of multiplexed flow cytometry are invaluable toclinical and research laboratories, and are used for a wide range ofapplications from studies of cellular biology to disease diagnosis.However, due to existing constraints placed by conventional methods andreagents, flow cytometry has almost universally been practiced usingsubjective analysis parameters. The quantitative approach to flowcytometry described herein provide researchers more flexibility inexperimental design and a streamlined approach to quantitation; thus,this is an important development in the field that addresses many of thecurrent challenges to conventional flow cytometry.

B. Single-Cell Mass Cytometry

Embodiments described herein may also be used to provide a quantitativeapproach to single-cell mass cytometry (CyTOF). CyTOF is anotherplatform that can be used to simultaneously analyze multiple parametersof individual cells in a sample (Bendall et al., Science, 332:687-696(2011)). The work flow is comparable to that of fluorescence flowcytometery. In general, antibodies labeled with heavy metals ortransition element isotopes are used to bind target epitopes on orwithin cells. The antibody-bound cells are then vaporized, such as byspraying single-cell droplets into an inductively coupled argon plasmaat approximately 5500 K. Vaporization induces ionization of the cellsatomic constituents. The elemental ions are then sampled by aTime-Of-Flight (TOF) mass spectrometer and quantified. The signal foreach metal/isotope that labeled a particular cell are thereby detected.

C. Antibody:Oligonucleotide Targeting Constructs

As discussed above, labeled antibodies are employed in both flowcytometry and CyTOF platforms. Although there are a variety ofcommercially available antibodies biochemically conjugated tofluorochromes, the majority of clones are only available in a limitednumber of colors, often in the same standard fluorochrome such asfluorescein. The interchangeable “Mix and Match” hybridization strategyof the antibody:oligonucleotide targeting constructs disclosed herein,offers a significant improvement over existing methods. In particular,antibody:ligand targeting constructs facilitate greaterinterchangeability than afforded using direct antibody-fluorophoreconjugates, and provide a more convenient solution for multiplexedlabeling that indirect labeling techniques based on biotin-streptavidinchemistry.

Antibodies are glycoproteins belonging to the immunoglobulinsuperfamily. Antibodies typically are made of two large heavy chains andtwo small light chains. There are several different types of antibodyheavy chains, and several different kinds of antibodies, which aregrouped into different isotypes (IgA, IgD, IgE, IgG and IgM in mammals)based on which heavy chain they possess. Though the general structure ofall antibodies is very similar, a small region known as thehypervariable region at the tip of the protein is extremely variable.This allows for enormous diversity of antibodies to recognize a widevariety of antigens.

The antibody portion of the antibody:ligand targeting construct maycomprise any immunologic binding agent, such as IgG, IgM, IgA, IgD andIgE or Fab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, and scFv(single chain Fv) fragments thereof. In certain aspects the antibody isa monoclonal antibody. Monoclonal antibodies (MAbs) may be readilyprepared through use of well-known techniques, such as those exemplifiedin U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically,this technique involves immunizing a suitable animal with a selectedimmunogen composition, e.g., a purified or partially purified protein,polypeptide, peptide or domain, be it a wild-type or mutant composition.The immunizing composition is administered in a manner effective tostimulate antibody producing cells. Following immunization, somaticcells with the potential for producing antibodies, specifically Blymphocytes (B cells), are selected for use in the MAb generatingprotocol. These cells may be obtained from biopsied spleens, tonsils orlymph nodes, or from a peripheral blood sample. The antibody-producing Blymphocytes from the immunized animal are then fused with cells of animmortal myeloma cell, generally one of the same species as the animalthat was immunized. Myeloma cell lines suited for use inhybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Typically, selection of hybridomas is performed byculturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like. Fragments of monoclonal antibodies can be obtainedby enzymatic digestion, cleavage, or chemical reduction of monoclonalantibodies. Alternatively, monoclonal antibody fragments may besynthesized using an automated peptide synthesizer or producedrecombinantly.

The antibody may be conjugated to a ligand using a variety oftechniques. One approach is the use of HyNic-4FB. Briefly,succinimidyl-6-hydrazinonicotinamide acetone hydrazone (S-HyNic) isadded to purified antibody, converting free amino groups on lysines nearthe antibody hinge region to HyNic moieties. Similarly,succinimidyl-4-formylbenzamide (S-4FB) added to amino-modified oligoconverts amino groups to 4-FB moieties. When combined in the presence ofaniline catalyst, the HyNic and 4-FB sites on modified biomoleculesreact to produce a stable, covalent hydrazone bond and forming theantibody:oligo conjugate. Following purification using a nickel column,this process results in >95% yield of antibody:oligo conjugate.

The antibody may alternatively be conjugated to a ligand according to avariety of bioconjugation techniques known to those in the art. Theseinclude modification of amine, carbonyl, hydroxyl, sulfhydryl, or otheravailable groups on biomolecules to incorporate linker moieties, withsubsequent reaction of the linker moieties to form a conjugate. Linkerpairs may include (strept)avidin-biotin, azide-acrylamide,thiol-maleimide, and others (Hermanson, Bioconjugate Techniques,Academic Press 1996). However, modification and linkage of biomoleculesmay affect biological activity of either the antibody and/or theoligonucleotide, so milder reactions proceeding at neutral pH,temperature and salt conditions (e.g., hydrazone chemistry) arepreferable to reactions requiring harsh conjugation conditions (e.g.,sulfhydryl reduction followed by thiol-maleimide modification).

Herein, a ligand generally comprises an oligonucleotide linked to anantibody, although alternative ligands (e.g. peptides or haptens) may beused. Oligonucleotides conjugated to the antibodies are designed tohybridize to complementary, labeled oligonucleotides. As used herein,“hybridization,” “hybridizes” or “capable of hybridizing” is understoodto mean the forming of a double- or occasionally triple-strandedmolecules, or a molecule with partial double or triple stranded nature.The term “anneal” as used herein is synonymous with “hybridize.” Animportant parameter for describing oligonucleotides and theirinteraction with complementary sequences is the so-called T_(m), thetemperature at which 50% of the nucleic acid duplex formed byhybridization of complementary sequences is dissociated. The T_(m)varies according to a number of sequence dependent properties includingthe hydrogen bonding energies of the canonical pairs A/U-T and G-C(often measured as the GC percentage or base composition), the stackingfree energy and, to a lesser extent, nearest neighbor interactions.These energies vary widely among oligonucleotides that are typicallyused in hybridization assays. For example, hybridization of two probesequences composed of 24 nucleotides, one with a 40% GC content and theother with a 60% GC content, with its complementary target understandard conditions theoretically may have a 10° C. difference inmelting temperature.

In multiplex assays, problems in hybridization occur when the hybridsare allowed to form under hybridization conditions that include a singlehybridization temperature that is not optimal for correct hybridizationof all oligonucleotide sequences of a set. Mismatch hybridization ofnon-complementary probes can occur, forming duplexes with measurablemismatch stability. Mismatching of duplexes in a particular set ofoligonucleotides can occur under hybridization conditions where themismatch results in a decrease in duplex stability that results in ahigher Tm than the least stable correct duplex of that particular set.For example, if hybridization is carried out under conditions that favorthe AT-rich perfect match duplex sequence, the possibility exists forhybridizing a GC-rich duplex sequence that contains a mismatched basehaving a melting temperature that is still above the correctly formedAT-rich duplex. Accordingly, methods of Tm normalization have beenemployed in an effort to maintain equivalent hybridization stringencybetween nucleic acids having disparate Tms. Some of these methodsinclude the use of non-natural nucleic acid backbones (LNA for example)or the use of hairpin probes.

Typically, it will be desirable that the oligonucleotides conjugated tothe antibody are not cross-reactive with other nucleic acids that may bepresent in a sample. And, in multiplexed application, it will also bedesirable that an oligonucleotide conjugated to one antibody is notcross-reactive with the labeled oligonucleotide probe for anotherantibody:oligonucleotide conjugate. There are a number of differentapproaches for selecting complementary oligonucleotide sequences for usein multiplexed hybridization assays. The selection of sequences that canbe used as zip codes or tags in an addressable array has been describedin the patent literature in an approach taken by Brenner and co-workers(U.S. Pat. No. 5,654,413, incorporated herein by reference). Inaddition, U.S. Pat. No. 7,226,737, incorporated herein by reference,describes a set of 210 non-cross hybridizing tags and anti-tags. U.S.Published Application No. 2005/0191625, incorporated herein byreference, discloses a family of 1168 tag sequences with a demonstratedability to correctly hybridize to their complementary sequences withminimal cross hybridization.

The nucleic acids disclosed herein may be prepared by any techniqueknown to one of ordinary skill in the art, such as for example, chemicalsynthesis, enzymatic production, or biological production. Non-limitingexamples of a synthetic nucleic acid (e.g., a syntheticoligonucleotide), include a nucleic acid made by in vitro chemicalsynthesis using phosphotriester, phosphite or phosphoramidite chemistryand solid phase techniques such as described in EP 266,032, incorporatedherein by reference, or via deoxynucleoside H-phosphonate intermediatesas described by U.S. Pat. No. 5,705,629, incorporated herein byreference. Various different mechanisms of oligonucleotide synthesishave been disclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PolymeraseChain Reaction (PCR) (see for example, U.S. Pat. No. 4,683,202 and U.S.Pat. No. 4,682,195, each incorporated herein by reference), or thesynthesis of an oligonucleotide described in U.S. Pat. No. 5,645,897,incorporated herein by reference. A non-limiting example of abiologically produced nucleic acid includes a recombinant nucleic acidproduced (i.e., replicated) in a living cell, such as a recombinant DNAvector replicated in bacteria (see for example, Sambrook et al., 2001).

The oligonucleotides may include nucleotide isomers or base analogs. Anucleic acid sequence may comprise, or be composed entirely of, ananalog of a naturally occurring nucleotide. Nucleotide analogs are wellknown in the art. A non-limiting example is a “Peptide Nucleic Acid,”also known as a “PNA,” “peptide-based nucleic acid analog,” or “PENAM,”described in U.S. Pat. Nos. 5,786,461, 5,891,625, 5,773,571, 5,766,855,5,736,336, 5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each ofwhich is incorporated herein by reference. PNAs generally have enhancedsequence specificity, binding properties, and resistance to enzymaticdegradation in comparison to molecules such as DNA and RNA (Egholm etal., 1993; PCT/EP/01219). Another non-limiting example is a LockedNucleic Acid or “LNA.” An LNA monomer is a bi-cyclic compound that isstructurally similar to RNA nucleosides. LNAs have a furanoseconformation that is restricted by a methylene linker that connects the2′-O position to the 4′-C position. Yet another non-limiting example isa “polyether nucleic acid,” described in U.S. Pat. No. 5,908,845,incorporated herein by reference. In a polyether nucleic acid, one ormore nucleobases are linked to chiral carbon atoms in a polyetherbackbone.

D. Oligospheres

Methods described herein can be applied to create quantitativeligand-surfaced microspheres using any type of ligand (e.g.oligospheres, peptides, haptens). However, various embodiments disclosedherein use oligospheres as quantitative reference standards.Oligospheres comprise microspheres conjugated to oligonucleotides. Insome embodiments, the oligonucleotides will be conjugated substantiallyuniformly to the entire surface of the oligosphere (as in FIG. 5A). Inother embodiments, the oligonucleotides will be conjugated at increasingtitrations to the surface of the oligosphere (as in FIG. 5B).

The oligonucleotides may be conjugated to the microspheres according toa variety of techniques known to those in the art. Similar toantibody-oligo conjugation, the oligonucleotide-microsphere conjugationprocedure may involve modification of amine, carboxyl, hydroxyl or otherreactive groups on oligonucleotides and microsphere surfaces in order toincorporate linker moieties for subsequent conjugation reactions; linkerchemistry may include HyNic/4FB (hydrazone), (strept)avidin/biotin,phosphoramidite, octadinyl dU, and other chemistries. Alternatively, themicrospheres may be pre-manufactured to present surface reactive groupsto which reactive-group bearing oligo may be conjugated (e.g., amino- orstreptavidin-modified microspheres). Typically, the oligonucleotideswill be conjugated substantially uniformly to the entire surface of theoligosphere. In certain aspects, a non-reactive spacer sequence isplaced between the microsphere and the region of the oligonucleotidethat is complementary to the probe. Such non-reactive spacers may, forexample, facilitate conjugation to the microsphere and/or reduce sterichindrance of the oligonucleotide. Examples of non-reactive spacersinclude Poly Ethylene Glycols (PEGs) or oligonucleotide domains designedfor minimal cross-reactivity (e.g. poly-Thymine, “PolyT”).

In certain embodiments, the oligospheres are hybridized to the samelabeled oligonucleotide probe that is used to hybridize to theantibody:oligonucleotide targeting construct. Thus, the oligospheres andthe cells in the assay are labeled with the same label. FIGS. 5A-5Billustrate two methods for preparation of a titrated population ofquantitative oligospheres. As shown in FIG. 5A, oligonucleotides areconjugated to microspheres at surface saturation. A complementaryoligo:fluorophore labeling construct is then added at increasing levelsof titration, creating populations of fluorescent microspheres ofincreasing signal intensity. Following oligo:fluorophore hybridization,remaining (free) surface oligonucleotide may be passivated by theaddition of unmodified complementary oligonucleotide to reducenonspecific reactivity of free oligo (data not shown). The populationsof fluorophore-hybridized microspheres are then mixed, and can then beadded to cells stained with antibody::fluorophore labeled targetinghybrid. As shown in FIG. 5B, oligonucleotides are conjugated tomicrospheres at increasing surface saturations, but are not yet labeledwith complementary oligo:fluorophore. They are first combined with cellsbearing targeting construct (i.e. antibody:oligo). Complementaryoligo:fluorophore labeling construct is then added in sufficient amountto label both cells and oligospheres.

Using either preparation method allows the mixed sample of cells andquantitative oligospheres to be analyzed by flow cytometry, with theoligospheres providing an internal standard curve for quantitation ofcellular ABC. Accordingly, the oligosphere data can be immediately andeasily used for straightforward ABC calculation as described herein.

E. Analysis of Cells

Flow cytometry and CyTOF are valuable tools for study of cells. Inparticular, multiplexed cellular phenotyping is a principal strategyapplied in immunology research. Surface antigens indicative of immunecell status are detected by multiplexed antibody labeling, the sample isanalyzed by flow cytometry or CyTOF, and phenotypic subsetidentification is conducted. Using data analysis software, subsets aregated for inclusion in or exclusion from further analysis.

Various embodiments disclosed herein, address various challengespresented by conventional fluorescence flow cytometric methods byutilizing a DNA-Directed Assembly (DDA) strategy for cellular labeling.Antibody:oligonucleotide targeting constructs are hybridized tocomplementary oligo:fluorophore labeling constructs in solution tocreate a labeled targeting hybrid. The antibody::fluorophore labeledtargeting hybrid is then used to probe cellular antigens.Fluorophore-hybridized microspheres added to the cytometric analysis areused to convert relative units of fluorescence to quantitative measuresof Labeling construct Per Event (LPE). LPE is then used to calculate thenumber of Antibodies Bound per Cell (ABC). This approach can also beadapted to CyTOF analysis by replacing the fluorophore with ametal/isotope label.

Antibody:oligonucleotide targeting constructs comprising antibodiesspecific to various immune cell surface antigens can be used inmultiplexed cellular phenotyping. Peripheral Blood Mononuclear Cells(PBMC) are comprised of cells of myeloid and lymphoid lineages. Myeloidcells include monocytes, macrophages, and dendritic cells. Lymphoidcells include T cells, NK cells, B cells, and lymphoid dendritic cells.The expression patterns of surface antigens in different immune celltypes are known to those in the art. A description of some of theseexpression patterns is provided below.

Natural Killer cells (NK cells) are a type of cytotoxic lymphocyte. NKcells are activated in response to interferons or macrophage-derivedcytokines, and they play a major role in the rejection of tumors andcells infected by viruses. NK cells are characterized by their lack ofthe T cell receptor (CD3) and their expression of CD56 on their surface.Accordingly, these characteristics may be used to separate NK cells fromother cell types.

T cells play a role in cell-mediated immunity. One way in which T cellscan be distinguished from other lymphocytes, such as B cells and NKcells, is by the presence on their cell surface of the T cell receptor.Activation of CD8+ T cells and CD4+ T cells occurs through theengagement of both the T cell receptor and CD28 on the T cell by theMajor Histocompatibility Complex (MHC) peptide and B7 family members onan antigen presenting cell. Activation-associated surface antigen CD43is expressed at distinct low and high levels, and lymphocyte homingmolecule CD62L is expressed at a range of levels as it is degraded uponcellular activation. Monocytes also express CD4, but they can bedistinguished from CD4+ lymphocytes, because monocytes also express CD14on their surface.

In some aspects of the invention, the cells are mammalian cells,including cultured mammalian cells (e.g., murine or human tumor, stem,or immortalized cell lines), cells derived from laboratory rodents, orcells derived from human patient samples such as whole blood,fine-needle cellular aspirates, or biopsy tissue. In certainembodiments, the cell sample is derived from an environmental samplesuch as a water, soil, or air. In other embodiments, the sample is froma plant, bacteria, virus, fungi, protozoan, or metazoan.

F. Kits

The present invention also provides kits. Any of the componentsdisclosed herein may be combined in a kit. In certain embodiments thekits comprise one or more of an targeting construct, a labelingconstruct, and/or ligand-surfaced microspheres.

In certain embodiments, the kit comprises a composition comprising atitrated population of labeled oligospheres, wherein the titratedpopulation of labeled oligospheres comprises at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 subpopulations of labeled oligospheres, wherein eachof the subpopulations of labeled oligospheres is hybridized to adifferent amount of labeling construct. In certain aspects, the titratedpopulation of labeled oligospheres are combined in a single container inthe kit. In other aspects, the subpopulations are provided of labeledoligospheres are provided in separate containers in the kit. In someembodiments, the kit comprises an antibody:oligonucleotide targetingconstruct and/or a fluorophore:oligonucleotide labeling construct. Incertain embodiments, the oligonucleotide in thefluorophore:oligonucleotide labeling construct is complementary to theoligonucleotides on the oligosphere and the antibody:oligonucleotidetargeting construct.

The kits will generally include at least one vial, test tube, flask,bottle, syringe or other container, into which a component may beplaced, and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional containers into which the additionalcomponents may be separately placed. However, various combinations ofcomponents may be comprised in a container. In some embodiments, all ofthe oligosphere subpopulations in a series are combined in a singlecontainer. In other embodiments, some or all of the oligospheresubpopulations in a series are provided in separate containers.

The kits of the present invention also will typically include packagingfor containing the various containers in close confinement forcommercial sale. Such packaging may include cardboard or injection orblow molded plastic packaging into which the desired containers areretained. A kit may also include instructions for employing the kitcomponents. Instructions may include variations that can be implemented.

G. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

1. Selection of oligonucleotide sequences, antibodies, and fluorophores

Oligonucleotide sequences are shown in Table 1. Oligo pairs 1/1′, 2/2′,3/3′, and 4/4′ were designed and validated by Feldkamp et al [7] to havelow reactivity with unmatched oligo sequences, high melting temperature,stable and robust hybridization activity, and desirable hairpinformation characteristics (i.e., retain hairpins at higher temperatures,reducing oligo crosstalk between unmatched sequences). Oligo crosstalkwas tested for pairs 1-4 by staining cells with a matrix of matched andunmatched antibody:oligo::oligo:fluor pairs, and observed undesirablecrosstalk to be <2% in all cases. This has implications for multiplexedcell labeling (i.e., any number of antibody:oligos can be mixed togetherwith oligo:fluors in solution without assay interference by crosstalkvia oligo exchange).

TABLE 1 Oligonucleotide Sequences Tm Oligo Sequence Bases (° C.) oligo-1CCTGCGTCGTTTAAGGAAGTAC 22 62.2 oligo-1′ GTACTTCCTTAAACGACGCAGG 22 62.2oligo-2 GGTCCGGTCATAAAGCGATAAG 22 62.2 oligo-2′ CTTATCGCTTTATGACCGGACC22 62.2 oligo-3 GCTGACATAGAGTGCGATAC 20 62.2 oligo-3′GTATCGCACTCTATGTCAGC 20 62.2 oligo-4 TGTGCTCGTCTCTGCATACT 20 63.5oligo-4′ AGTATGCAGAGACGAGCACA 20 63.5 oligo-A GGAAGCGGTGCTATCCATCT 2071.1 oligo-A′ AGATGGATAGCACCGCTTCC 20 71.1 oligo-1 (SEQ ID NO: 1),oligo-1′ (SEQ ID NO: 2), oligo-2 (SEQ ID NO: 3), oligo-2′ (SEQ ID NO:4), oligo-3 (SEQ ID NO: 5), oligo-3′ (SEQ ID NO: 6), oligo-4 (SEQ ID NO:7), oligo-4′ (SEQ ID NO: 8), oligo-A (SEQ ID NO: 9), oligo-A′ (SEQ IDNO: 10)

In addition to oligo pairs 1/1′-4/4′, novel oligo pair A/A′ was designedto have similar desirable qualities to the Feldkamp oligos using CANADADNA sequence generating software (Feldkamp, et al., 2002; Feldkamp, etal., 2010) to simulate hybridization, melting and folding activity.Oligo-A/A′ was used as a “universal” oligo sequence (see discussionfollowing).

Antibodies were selected targeting commonly-probed T-cell markers CD4and CD8, as well as activation-associated surface antigen CD43, which isexpressed at distinct low and high levels, and lymphocyte homingmolecule CD62L, which expresses at a range of levels as it is degradedupon cellular activation. Antibody clones were chosen based onpreviously validated activity for αCD4 (clone GK1.5), αCD8 (2.43.1),αCD43 (S7) and αCD62L (MEL-14) (activity confirmed by personalcommunication). As a panel, these four antibody targets allow forphenotypic delineation of several subsets of murine T-lymphocytes.Antibody-oligo conjugates are listed in Table 2.

TABLE 2 Oligonucleotide conjugates Ig:oligo conjugate Clone Oligos perIg Conjugate DOL αCD4:oligo-1 GK1.5 2.1 oligo-1′:Dy490 4.7 αCD8:oligo-22.43.1 2.4 oligo-2′:Dy549 6.4 αCD43:oligo-3 S7 3.1 oligo-3′:Dy649 8.1αCD62L:oligo-4 MEL-14 2.6 oligo-4′:Dy405 10.4 αCD4:oligo-A GK1.5 4.6oligo-A′:Dy490 7.6 αCD8:oligo-A 2.43.1 2.8 oligo-A′:Dy549 6.5

Oligo:fluorophores labeling constructs used for this study are alsodescribed in Table 2. DyLight fluorophores were chosen due to theirsuitability for 4-laser flow cytometry, relatively narrowexcitation/emission spectra which reduces or eliminates the need forspectral compensation of multiplexed staining data, and availability inNHS-ester modified format for conjugation to oligo-dextran scaffolds(see below).

2. Oligonucleotide Conjugate Preparation

Antibody:oligonucleotides targeting constructs were prepared as shown inFIG. 1A. Briefly, succinimidyl-6-hydrazinonicotinamide acetone hydrazone(S-HyNic) was added to purified antibody, converting free amino groupson lysines near the antibody hinge region to HyNic moieties. Similarly,succinimidyl-4-formylbenzamide (S-4FB) was added to amino-modified oligoconverts amino groups to 4FB moieties. When combined in the presence ofaniline catalyst, the HyNic and 4-FB sites on modified biomoleculesreact to produce a stable, covalent hydrazone bond and forming theantibody:oligo conjugate. Following purification using a nickel column,this process resulted in >95% yield of antibody:oligo targetingconstructs.

The preparation of oligo:fluorophore labeling constructs is shown inFIG. 1B. Amino-dextran bearing ˜20 amino groups per dextran was firstHyNic-modified using a limited amount of S-HyNic to result in 3-4 HyNicmoieties per dextran. To the HyNic-amino-dextran was added astoichiometrically limiting amount of 4-FB-oligo such that the number ofoligos per dextran in the final product was limited to ≦1, an importantfactor necessary to restrict oligo hybridization at a 1:1 ratio ofantibody:oligo targeting construct to oligo:fluor labeling construct.Multiple-oligo hybridization would result in more than one antibody perdextran conjugate, which could produce unwanted double-hybridization andaggregation of conjugates.

Following oligo-coupling to the dextran scaffold, free amino groups onthe dextran remain available for reaction with NHS ester fluorophore(here, NHS-DyLight fluors were used). A molar excess of NHS-fluor wasadded to oligo-dextran, allowed to react and the final conjugate wascharacterized after desalting by dialysis. Characterization by A260assay allowed calculation of fluorophore Degree Of Labeling (DOL) of theconjugate. Oligo:fluorophore labeling constructs having DOL fromapproximately 3-15 fluors per dextran were prepared, with a finalconjugate yield of 15-20%.

Oligo-conjugates were utilized for cellular antigen labeling asillustrated in FIG. 1C. First, antibody:oligo targeting constructs werehybridized to complementary oligo:fluorophore labeling constructsbriefly in solution. The prepared antibody::fluorophore labeledtargeting hybrid was then used to label cells in the manner of aconventionally prepared antibody-fluorophore conjugate. Hybridizedlabeling constructs can be used to label cells for a single antigen, or(as shown in the figure), combined into a labeling cocktail formultiplexed cell labeling.

3. Optimization of Hybridization and Cell Labeling Conditions

A model system including freshly prepared normal B6 murine splenocytes,commonly used control and validation T-cell marker antibody CD4, andDyLight 490 (Dy490) fluorophore was used to determine optimal assayconditions for labeling-construct hybridization and viable cell stainingAntibodies and dextran-coupled fluorophores were oligo-modified aspreviously described. Cells were stained with antibody::fluorophorelabeled targeting hybrids in a conventional manner (e.g., added to Fcreceptor-blocked cells for 30 minutes at 4° C.), washed and analyzed byflow cytometry; CD4 staining was visualized for the gated lymphocytepopulation.

It was first investigated whether antibody:oligo targeting constructscould be hybridized to complementary oligo:fluorophore labelingconstructs briefly in solution, and the resulting solution of labeledtargeting hybrids then used to label cells. To this end, it washypothesized that the ratio of oligo:fluorophore labeling constructadded to antibody:oligo targeting construct would affect hybridizationin solution, and subsequently alter cytometric staining distribution oflabeled cells. In order to test this, a titration of increasing molarequivalents of oligo:fluorophore labeling construct was added to a fixedamount (6 pmol) of antibody:oligo targeting construct, from 0.5-10 molarequivalents (FIG. 2A). For antibody:oligo targeting construct having anMSR of ˜2 oligos per Ig molecule, 0.5 molar equivalents oligo:fluorlabeling construct represents the addition of 1 oligo:fluor labelingconstruct per Ig:oligo targeting construct. Results showed thepopulation of CD4+ cells to be similar for all titrations; however,nonspecific background staining caused by addition of excess fluorophoreincreased with addition of >1 molar equivalent oligo:fluor labelingconstruct. A titration of 0.5 molar equivalents oligo:fluor labelingconstruct was used for subsequent CD4 staining, and for antibodieshaving varying degrees of oligo-modification, equivalents were addedlimiting hybridization to one oligo:fluor labeling construct perIg:oligo targeting construct (i.e., if Ig:oligo MSR ˜4, then 0.25equivalents oligo:fluor were added).

It also was investigated whether cells could be first labeled withantibody:oligo targeting construct, and then hybridized with oligo:fluorlabeling construct in situ (FIG. 2B). Equal amounts of antibody andfluorophore oligo-conjugates were used for the two approaches, and cellstaining and analysis conditions were identical. Results showed celllabeling via in situ hybridization to be effective, and very similar tolabeling via hybridization in solution.

To confirm that CD4+ staining was indeed antigen-positive labeling andnot an artifact of nonspecific oligo binding, hybridization ofoligo:fluor labeling construct was blocked by hybridizing CD4antibody:oligo targeting construct to a “blocking” oligo sequencecomplementary to the anti-CD4-oligo at the 5′ end, with a sequence(oligo-4) unmatched to oligo:Dy490 at the 3′ end. Followinghybridization of the blocking oligo, oligo:Dy490 was applied. Theblocked construct was applied to cells, and the cells were analyzed vscells stained with unblocked, prehybridized xCD4::Dy490 labeledtargeting hybrid (FIG. 2C). Results showed the blocking oligo (bluehistogram) effectively prevented hybridization of the oligo:Dy490labeling construct; no CD4+ population was evident, whereas celllabeling with the unblocked construct clearly resulted in a distinctCD4+ peak. The high background level in both samples was due to theexperimental conditions, in which a 5-fold molar excess of oligo:Dy490labeling construct was applied in order to fully test the ability of theblocking oligo at saturating conditions. The blocking oligo did notprevent nonspecific binding of the oligo:Dy490 labeling construct atthis level of saturation, leading us to conclude that it is thedextran:fluor that is responsible for the nonspecific signal. However,this issue can typically be avoided by hybridizing at the optimizedtitration of 0.5 molar equivalents.

Hybridization has been well-described to be both time andtemperature-dependent. A range of hybridization times from 15-60 minuteswith incubation at 4° C., room temperature (24° C.), or 37° C. (FIG. 2D)were tested. Results showed a clear CD4+ signal at all time andtemperature conditions tested, with negligible variance. The sequencesselected for this study were designed to have high melting temperatures(Tm) and specific and stable hybridization activity, as previouslyreported by Feldkamp et al.

A further optimization test was designed to determine whether the numberof fluorophores per dextran scaffold (Degree Of Labeling, DOL) affectedsignaling of labeled cells (FIG. 2E). Oligo-dextran-Dy490 conjugateshaving approximately 3-15 Dy490 per dextran were prepared. Theseconjugates were hybridized to anti-CD4:oligo targeting construct aspreviously described. Results showed optimal signaling distribution atDOL ˜7, with a decrease in positive-peak resolution at DOL <7 and amarked decrease in positive-peak median fluorescence intensity (GMFI) atDOL >7, most likely due to fluorescence self-quenching occurring as aresult of spatial proximity of fluorophores added in excess to thedextran scaffold. Testing of additional fluorophores indicated that fordimmer fluors (e.g., Dy405), a higher degree of labeling is optimal (DOL˜10; data not shown).

In summary, the hybridization-labeling assay is relatively robust. Molarequivalents of oligo:fluor labeling construct are preferably limited to≦1× relative to the amount of antibody:oligo targeting construct.Oligo-conjugates can be hybridized either in solution or in situ forspecific and effective labeling of cells. With these particular oligosequences, a wide range of time and temperature conditions can beemployed without significant variation in construct activity. Target DOLshould be approximately 7 fluors per dextran, but a range of DOL'sprovide adequate labeling of antigen-positive cell populations.

4. Multiplexed Antigen Labeling

Using optimized assay conditions, four labeled targeting constructs werethen prepared and used to label cells for a single antigen, or combinedinto a multiplexed labeling cocktail to label a single cell sample forfour antigens at once (FIG. 3A). For these tests, a panel ofoligo-conjugated antibodies against T-cell markers CD4 and CD8,activation-associated antigen CD43, and lymphocyte homing molecule CD62Lwas used. Each antibody:oligo targeting construct was hybridized tocomplementary oligo:fluorophore labeling construct in solution, usingthe Dylight fluors Dy490, Dy549, Dy649, or Dy405. The antibody::fluorlabeled targeting hybrids were then used to label normal B6 murinesplenocytes and the stained cells were analyzed by flow cytometry. Allantibody-labeled cell samples displayed clearly evident antigen-positivepopulations. Positive-labeled cell populations were within expectedranges [10-14]. Multiplexed staining was comparable to single-antigenstaining; cells stained with fluorophore-only exhibited varying degreesof nonspecific staining when compared to unstained controls, fromnegligible (Dy490) to moderately high (Dy649).

Multiplexed staining results displayed as 2-channel, 2D dot plotsallowed for phenotypic delineation of the cell population (FIG. 3B).Lymphocytes are displayed as CD4 vs CD8 (panel i), CD43 (panel ii),CD62L (panel iii), or gated on the CD4+ population and displayed on aCD43 vs CD62L 2D plot (panel iv). In each panel, cellular subsets aredistinctly evident; for example, CD4+ and CD8+ T-lymphocytes are clearlydefined (30% and 27% respectively); populations of CD43^(HIGH)lymphocytes are visible for CD4− and CD4+ cells (34%, 7%); and twodistinct CD62L+ groups are evident, either CD4− (55%) or CD4+ (20%).Lymphocytes displayed as CD8 vs CD43 (panel v) or CD62L (panel vi) showclear double-stained populations in both plots. CD43 vs CD62L (panelvii) also shows double-stained cells (58%), with CD43^(HIGH) CD62L+cells representing 33% of total lymphocytes. Finally, gated CD8+lymphocytes are almost entirely (93%) triple-positive for CD8+ CD43+CD62L+. These results provide substantial evidence that theoligo-conjugates can be used for specific and sensitive multiparametercellular phenotyping.

5. Interchangeable Fluorophore Hybridization Using the Universal OligoSequence

For experiments discussed thus far, the oligo sequences 1/1′-4/4′ wereused to hybridize antibodies with fluorophores. However, by utilizing asingle oligo pair (A/A′) conjugated to either antibodies (e.g., anIg:oligo-A targeting construct) or dextran:fluors (e.g., anoligo-A′:fluor labeling construct), any antibody may easily behybridized to any fluorophore. This “mix and match” approach isillustrated in FIG. 4A. Cytometric data obtained using CD4 and CD8antibodies hybridized to four fluorophores validated this approach, asantigen-positive staining was very similar across fluorescent channelsfor both antibodies (FIG. 4B).

However, utilizing one oligo pair for all constructs potentially posed aproblem for multiplexing, i.e when constructs are combined, freeoligo:fluor labeling constructs could hybridize to any antibody:oligotargeting construct, or oligo:fluor labeling constructs coulddehybridize and exchange. To test this, CD4/CD8 double staining wasperformed using no blocking methods to evaluate unwanted crosstalk.Indeed, crosstalk was observed at 1-5%, with the highest levels measuredafter the double-staining solution was left overnight at roomtemperature. The inventors hypothesized that a saturating amount ofunmodified oligo would successfully outcompete free oligo:fluor labelingconstruct for binding sites, thus preventing crosstalk. To test this,the inventors added unmodified oligo-A at increasing saturations toprehybridized antibody::fluorophore labeled targeting hybrid from 0-100molar equivalents blocking oligo-A. The ‘blocked’ constructs were thenmixed and used to stain cells. Results indicated that crosstalk, whichwould be evident in the double-positive quadrants, was reduced to ˜0.5%by the addition of 40× equivalents blocking oligo (FIG. 4C). CD4+ andCD8+ populations are clearly seen in both plots, either CD4::Dy490 (FIG.4C(i), lower right cluster) vs CD8::Dy649 (FIG. 4C(i), upper leftcluster), or CD4::Dy405 (FIG. 4C(ii), lower right cluster) vs CD8::Dy549(FIG. 4C(ii), upper left cluster).

6. Quantitation Using Oligonucleotide-Coated Particles

Two methods of preparation of quantitative fluorophore-hybridizedoligospheres are illustrated in FIG. 5A-5B: Method I, “parallellabeling” and Method II, “combined labeling”.

In Method I (FIG. 5A, “parallel labeling”), linker-modified paramagneticmicrospheres are conjugated with a saturating amount of linker-reactiveoligonucleotide, resulting in oligo-conjugated microspheres. Theoligospheres are then hybridized to complementary oligo:polyfluorlabeling constructs at several levels of surface saturation, and thelabeled fluorophore-hybridized oligospheres are added to cellspreviously stained with the same labeling probe(s) for quantitation ofAntibody Binding per Cell (ABC). Thus, oligospheres and cells arelabeled separately, hence the term “parallel labeling”.

In Method II (FIG. 5B, “combined labeling”), linker-modifiedparamagnetic microspheres are conjugated with increasing titrations oflinker-reactive oligonucleotide, resulting in oligo-conjugatedmicrospheres of increasing oligo surface saturation. The oligo-surfacedmicrospheres are then combined with cells that have been labeled withantibody-oligo targeting construct that bears the same oligo sequence asthe oligospheres. To the cell-sphere mixture is then added an amount oflabeling construct sufficient to label both cells and oligospheres.Thus, oligospheres and cells are labeled together, hence the term“combined labeling”. Following combined labeling, the cell-spheremixture is analyzed for quantitation of ABC.

After preparation of fluorophore-labeled oligospheres using eitherMethod, the number of Labeling construct Per oligosphere Event (LPE) foreach saturation level must be determined by fluorimetric analysis (FIG.5C). LPE is a critical value for determination of Antibody Binding perCell (ABC). LPE is determined by measuring fluorescence of a populationsof oligospheres in wells of a microplate vs a standard curve of labelingconstruct in solution in the same microplate. Then, the precise numberof microspheres per sample is counted using a handheld particleanalyzer. These two measurements allow determination of LPE by [(mollabel per sample X (6×1023) molecules per mol)/number of oligospheresper sample]. The fluorometrically-determined LPE values of thequantitative oligospheres are recorded and later used to determine ABCfollowing cytometric analysis (example cytometric data shown in FIG.5D).

The ABC quantitation method was testing using four antibody-fluorophorepairs (CD4/Dy490; CD8/Dy549; CD43/Dy649; and CD62L/Dy405), with matchingfluorophore-hybridized oligospheres.

For quantitation by cytometric analysis, the quantitative microsphereswere added to an equal volume of viable murine splenocytes multi-stainedwith a panel of the same oligo:polyfluor labeling constructs (Method I,parallel labeling). The heterogeneous samples of cells and microsphereswere cytometrically analyzed (FIGS. 6A-6D). Cytometric analysis oflabeled cells and oligospheres results in cytometric fluorescence datafor antibody-stained cells along with an internal quantitative standardcurve provided by the oligospheres. The standard curve generated by theoligospheres is used to calculate quantitative ABC from arbitrary unitsof cytometric Geometric Mean Fluorescence Intensity (GMFI).

To create quantitative plots for each antibody/fluorophore pair, logGMFI values for each microsphere peak in each channel (see FIGS. 6A-6D)were calculated using FlowJo analysis software. As shown in graphs(FIGS. 7A-7D), log GMFIs were plotted against log LPE for each label(Dy490, Dy549, Dy649, Dy405), which had been determined by fluorimetricassay as described above. An exponential trendline was fit tomicrosphere data as shown.

Determination of ABC in the system is based on the assumption that oneoligo-polyfluor labeling construct is hybridized per antibody when alimiting amount of labeling construct is applied duringantibody::fluorophore oligo-construct hybridization. In other words, a1:1 ratio of label to antibody is assumed; therefore, the number ofoligo-polyfluor Label Per Event (LPE) is equal to number of AntibodiesBound per Cell (ABC). That is, [LPE=ABC]; and so ABC for cellular eventscan thus be calculated using the trendline equations shown in FIG. 7.Mean ABC can be calculated using the GMFI of a population of cellularevents (as noted in FIG. 7), or single-cell ABC can be calculated usingfluorescence intensity signal of any single cell recorded by thecytometer (FIG. 8).

Method II (combined labeling) was also conducted using CD4antibody:oligo targeting construct with a complementary oligo:AlexaFluor 488 labeling construct. (FIG. 9). Oligospheres at increasingsurface oligo saturation (0-100%) were combined with murine splenocytesbearing CD4 antibody:oligo targeting construct. Labeling construct wasthen applied at 2-fold excess to targeting construct (mol oligo/oligo)and the combined cell-oligosphere labeled sample was cytometricallyanalyzed. Oligospheres and lymphocytes were scatter gated (FIG. 9A) andCD4+ lymphocytes were gated using FL1 (Alexa 488) vs SSC (FIG. 9B). Thegated oligospheres and CD4+ lymphocytes were displayed on a histogramshowing fluorescence signal distribution of each population. Becausequantitation of ABCCD4 using these data would proceed exactly accordingto the methodology described above, the inventors did not recapitulatequantitation using these data.

ABC quantitation as described above was validated by head-to-headquantitation of ABC_(CD4) with commercially available PE-conjugated CD4antibody and PE quantitation microspheres (BD QuantiBrite PE, FIG. 10A).A specific monoclonal antibody was chosen for quantitation using bothsystems (clone GK1.5). Commercial quantitation was performed accordingto manufacturer protocol resulting in the graph and ABC trendlineequation shown in FIG. 10B. The commercial quantitation method was verysimilar to that performed using the oligosphere method described above,i.e., analyzing fluorescent microspheres and stained cells, plotting logfluorescence units vs known LPE, and converting fluorescence units toABC based on trendline using an assumption of 1:1 label:protein ratio.Results show oligosphere-based quantitation of ABC_(CD4) was verysimilar to ABC_(CD4) obtained using commercial microspheres (28.8×103 vs29.7×103 per cell).

Flowchart algorithms (FIGS. 11A-11B) depict a workflow for plannedcomputer analysis software that will be used to simplify and automatethe ABC quantitation methods described above. The software will utilizeinstrument-generated cytometer raw data (e.g., .fcs listmode files) tostreamline the various ABC quantitation procedures described above,using two Algorithms.

Algorithm I (FIG. 11A) accomplishes gating of oligospheres, calculationof gate GMFIs, and plots ABC quantitation standard curve withfluorescence-to-ABC conversion trendline. Algorithm II (FIG. 11B) thenanalyzes user-defined cellular events using the ABC quantitationcurve(s) generated by Algorithm I to convert arbitrary cellularfluorescence data to quantitative ABC data. ABC data for large cellularpopulations (thousands to millions of single events) can then bestatistically analyzed and/or displayed graphically by the user. Inearly versions the software will likely be spreadsheet-based, followedby increasingly advanced, user-friendly platforms as softwaredevelopment progresses.

7. Spectral Compensation Using Fluorophore-Hybridized Oligospheres

Spectral compensation, a common practice in multicolor cytometricanalysis, refers to the unmixing of overlapping fluorescent emissionspectra in effort to separate each color during analysis, thus enablingaccurate signal analysis in each antibody-specific fluorescent channel.

To validate oligospheres for use in spectral compensation (FIGS.12A-12C), oligospheres were hybridized to fluorescent oligo labelingconstructs having similar excitation and emission spectra (FL1, AlexaFluor 488; and FL2, Alexa Fluor 532). Oligospheres were prepared asdescribed and fluorescent oligo labeling constructs were commerciallyobtained (Integrated DNA Technologies). Single-fluorophore-hybridizedoligospheres were analyzed separately as compensation controls, and thenmixed ˜1:1 into a two-colored sample to which compensation controls wereapplied for spectral unmixing. Nonfluorescent microspheres were includedin the analysis as a negative control. A cytometer with somewhat limitedspectral capabilities (BD LSRII, 488 nm blue laser with FL1 & FL2detectors on shared laser line) was used for the analysis in order toprovide maximum necessity for spectral compensation.

Compensation was accomplished using typical methodology and analysissoftware (TreeStar's FlowJo Compensation Wizard). Single-coloroligosphere controls were recognized by software and auto-gatedappropriately (FIG. 12A; shown are singlet gates, positive gates,negative gates). An algebraic compensation matrix was calculated by thesoftware (not shown), and the matrix algebra was then applied by thesoftware to correct the two-color sample. The uncompensated two-colorsample (FIG. 12B) appeared to contain two populations both positive forFL1 and FL2, which was not the case; each sample was labeled with onlyone type of fluorophore, either FL1 or FL2. The compensated sample (FIG.12C) correctly depicts the distinct single-color FL1+ and FL2+populations, as well as the negative population. The low-level signaling(0-10³) of the negative population is caused by autofluorescence of themicrospheres.

8. Fluorophore-Hybridized Oligospheres for Cytometer Alignment andCalibration

A common application for fluorescent microspheres is the routinealignment and calibration of cytometer optical components.Fluorophore-hybridized oligospheres were evaluated for alignment andcalibration purposes as compared to commercially available fluorescentmicrospheres (FIGS. 13-14).

To evaluate whether oligospheres could potentially be used forinstrument alignment (FIG. 13), fluorophore-hybridized oligospheres wereprepared in a method resulting in single-fluorophore microspheres in avariety of spectral ‘colors’ (similar in concept to commerciallyprepared alignment microspheres, e.g., Spherotech Fluorescence AlignmentParticles). Oligospheres were hybridized with a saturating amount ofcomplementary oligo:fluorophore labeling constructs in distinct spectra(Alexa Fluor 488, 532, and 647). A single type of fluorophore washybridized to each sample of oligospheres.

The oligospheres (FIG. 13A) and commercial microspheres (FIG. 13B) werethen analyzed on a conventional cytometer to determine size distribution(scatter plots) and fluorescent signal (single-peak histograms).Post-acquisition, singlets were gated and CVs were determined forfluorescent signal histograms using analysis software. CVs were comparedfor oligospheres vs commercial microspheres in three fluorescentchannels (FL1, FL2, FL3).

Lower CVs are desirable for properly evaluating instrument alignment.Commercial microspheres had slightly lower CVs. The inventorshypothesize that the higher CVs seen with oligospheres is a result ofgreater size and granularity (FSC and SSC) distribution of theparticular microspheres used for this test (see scatter plots FIG. 13Avs 13B). A wide variety of microspheres can be used to prepareoligospheres; by adjusting the type of microsphere used, the inventorshope to reduce scatter variation, thereby reducing oligosphere CVs to alevel competitive with (or better than) current state-of-the-artalignment aids.

To evaluate whether oligospheres could potentially be used forinstrument calibration (FIG. 14), fluorophore-hybridized oligosphereswere prepared in a method resulting in microspheres in a variety ofincreasing intensities (similar in concept to commercially preparedalignment microspheres, e.g., Spherotech Calibration Particles).Oligospheres were hybridized with titrated amounts of complementaryoligo:fluorophore labeling construct (Alexa Fluor 488). A specifictitration was hybridized to each sample of oligospheres and then theoligospheres were mixed into a single batch for analysis (FIG. 14A). Theoligospheres were compared to commercially prepared microspheres (FIG.14B). Results showed distinct peak formation with a dynamic range ofsignaling comparable to commercial microspheres. For future development,additional peaks can be included in the oligosphere mixture, (e.g., 6-8peaks) and additional fluorophores can be used to test signaling acrossmultiple fluorescent channels (e.g., FL2, FL3, etc).

9. Discussion

Microsphere-based methods for flow cytometry enable both instrumentQuality Control (QC) via alignment and calibration, and cellularanalysis via Quantitative Flow Cytometry (QFC).

The novel oligosphere-based method enables cost-effective,spectrally-matched QC using oligospheres and labeling probes. Theinventors envision QC reagents will be provided with multiplexedantibody labeling kits, or as standalone QC products, either of whichthe inventors hope will encourage and improve routine QC acrossacademic, clinical, and industrial research laboratories.

Variation in the number of antigens per cell can indicate cellularphenotype, differentiation, and activation state, which makesmicrosphere-enabled quantitative flow cytometry useful as a researchtool and as a clinical diagnostic test (Hultin, et al., 1998; Lin, etal., 1998; Schlenke, et al., 1998). There are a variety of commercialcalibrants presently available to aid in QFC, yet there remainsignificant challenges. (as reviewed in Gratama, et al., 1998; Maher, etal., 2005 and discussed further below).

In addition to improved QC and QFC, the system offers more chromaticflexibility for day-to-day cytometry applications. The chromaticallyinterchangeable “Mix and Match” hybridization strategy (FIGS. 4A-4C)offers a significant improvement over existing methods, and thefluorophore-hybridized oligospheres enable fast and accurate spectralcompensation (FIGS. 12A-12C).

The “Mix and Match” strategy enables antibodies to quickly (minutes) belabeled with any fluorophore desired, a functionality that is extremelylimited in today's laboratories which often rely entirely on prelabeledfluorescent antibodies. In contrast to existing methods, the inventorshave shown that the system can be multiplexed using at least fourtargets, with the potential limiting factors being imposed only by thefluorophore-resolving capabilities of the cytometer being used (alimitation which similarly constrains conventional multiplexing).

The inventors envision software to be designed for automated, rapid QC,QFC, compensation, and cellular analysis using the system. Software willbe designed to incorporate techniques and on-screen tools familiar tothose of ordinary skill in the art. An example of an algorithm for QFCis shown in FIGS. 11A-11B, and other algorithms are envisioned to besimilarly designed. Software may be designed to be used with on-boardacquisition software (e.g., BD FACSDiva), post-data analysis software(e.g., TreeStar FlowJo), or for in-depth statistical analysis ofquantitative data, be spreadsheet-based.

In summary, the inventors envision the oligonucleotide-based system tobe an all-in-one solution to many of the challenges presented by currentflow cytometry methodologies, from day-to-day instrument maintenance toadvanced, quantitative cellular analysis.

10. Methods

a. Antibodies

Purified monoclonal antibodies for oligo-conjugation against murine CD4(clone GK1.5) or CD8 (clone 2.43.1) were obtained from the Frank W.Fitch Monoclonal Antibody Bank at University of Chicago (Chicago, Ill.).Monoclonal murine anti-CD43 (clone S7) was a gift from Dr. John Kemp atUniversity of Iowa School of Medicine (Iowa City, Iowa). Anti-CD62L(clone MEL-14) was obtained from American Type Culture Collection (ATCC;Manassas, Va.). Commercially prepared anti-CD4 (clone GK1.5,phycoerythin conjugate) was obtained from Becton Dickinson (BD; SanJose, Calif.).

b. Fluorophores

For oligo:polyfluor labeling constructs, four NHS-ester ‘DyLight’fluorophores (Dyomics, Germany) were selected for poly-conjugation tooligo-dextran scaffolds, including Dy490 (ex/em 490/516 nm; similar toFluorescein/FITC); Dy549 (ex/em 560/575; similar to R-Phycoerythrin/PE);and Dy649 (ex/em 655/676 nm; similar to allophycocyanin/APC); and Dy405(ex/em 400/420 nm; similar to Pacific Blue). For oligo:unifluor labelingconstructs, three Alexa Fluor fluorophores (488, 532, 647) were selectedand commercially conjugated (IDT, Coralville, Iowa) to oligonucleotidesequences.

c. Oligonucleotides

Four trial oligonucleotide pairs were selected from a previouslyvalidated sequence library developed by Feldkamp, et al., 2004;Feldkamp, et al, 2002. A unique “universal” oligonucleotide pair wasgenerated using DNA sequence generation and evaluation software.Oligonucleotides used for conjugation to antibodies ordextran-fluorochrome scaffolds (polyfluors) were commerciallysynthesized with an amino-C6 group at the 5′ end (Eurogentec, San Diego,Calif.). Oligonucleotides having single fluorophore molecules(unifluors) were commercially synthesized conjugated to fluorophores(IDT, Coralville, Iowa)

d. Microspheres

Commercialized fluorescent microspheres were used for ABC quantitation(BD, San Jose, Calif.) and for alignment and calibration examples(Spherotech, Glen Ellyn, Ill.) Microspheres used for oligo-conjugationwere 4FB-functionalized 3 μm paramagnetic particles (Solulink, SanDiego, Calif.).

e. Conversion of Amino-Oligonucleotides to 4FB-Oligonucleotides

5′-(C6-amino) oligonucleotides were dissolved in 500 μL ModificationBuffer (MB; 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) andtransferred to 3 kDa MWCO VivaSpin 500 diafiltration devices (SartoriusStedim Biotech, France). The oligo samples were centrifuged at 14,000×gfor approximately 15 minutes until the retentate volume was reduced to50 μL. Fresh MB (450 μL) was added to each sample and thoroughly mixedby pipet. This process was repeated a total of 4 times to completelyremove amine-containing salts carried over from oligonucleotidesynthesis. Finally, oligonucleotide samples were adjusted toapproximately 0.5 OD260/μL in preparation for modification.

A solution of Succinimidyl 4-FormylBenzoate (S-4FB; Solulink, San Diego,Calif.) was prepared at 50 mg/mL in anhydrous DiMethylFormamide (DMF)and added to each oligo sample at a 20-fold excess (mol) to ensurecomplete reaction. Reactions proceeded at room temperature (˜24° C.) for2 hours before being diluted to 500 μL with Conjugation Buffer (CB; 100mM sodium phosphate, 150 mM sodium chloride, pH 6.0). Excess S-4FB wasremoved via 4 rounds of diafiltration as described previously using CB.Post-modification oligo concentrations were adjusted to approximately0.3 OD260/μL.

f. Preparation of Antibody-Oligonucleotide Targeting Constructs

Antibodies were supplied in PBS at approximately 1 mg/mL based onNanoDrop A280 readings using an E1% value of 14.0. Antibodies weregently concentrated to 3-4 mg/mL using 30 kDa MWCO VivaSpin 500diafiltration devices. Antibodies were buffer exchanged into MB via 2 mL‘Zeba’ desalting columns (Thermo Scientific, Rockford, Ill.) and proteinconcentration determined by A280. Proteins were subsequently modifiedwith the chemical crosslinker Succinimidyl 6-HydraziNicotinate acetonehydrazone (S-HyNic; Solulink, San Diego, Calif.) at a 20-fold excess oflinker to protein (mol). Following incubation for 2 hours at 24° C., theantibodies were liberated of unreacted linker by desalting with 2 mLZeba columns equilibrated in CB. Molar Substitution Ratios (MSRs) ofincorporated HyNic to antibody were determined via 2-SulfoBenzaldehyde(2-SB) assay using a molar extinction coefficient of 28,500 L mol-1 cm-1for the hydrazone at λmax=350 nm. MSR values ranged from 6-8 HyNic perantibody molecule.

To the HyNic-modified antibodies in conjugation buffer were added 4equivalents (mol) of 5′-4FB-modified oligonucleotide followed by 10%(v/v) of TurboLink catalyst (100 mM aniline, 100 mM sodium phosphate,150 mM sodium chloride, pH 6.0; Solulink, San Diego, Calif.). Theantibody-oligo conjugation reaction was allowed to proceed overnight at4° C. Excess oligonucleotide was removed from the conjugated product bysize exclusion chromatography using an HR-10/30 Superdex 200 PG column(GE Healthcare, Piscataway, N.J.). Removal of free oligonucleotide fromthe conjugated product was complete as evidenced by baseline resolutionof the two A260 peaks. MSR values for oligos/antibody were determined bythe ratio of the area under the A260 curves. Final protein concentrationof the conjugates was determined by BCA protein assay (ThermoScientific, Rockford, Ill.).

g. Preparation of Dextran-Oligonucleotide Heterodimers

A 1:1 oligo:dextran conjugate was prepared using the followingprocedure: 70 kDa amino-dextran containing approximately 20amines/dextran (Invitrogen, Carlsbad, Calif.) was dissolved inmodification buffer at 11.5 mg/mL and desalted into the same buffer viaa 5 mL Zeba column to remove traces of amine-containing contaminants.The dextran solution was treated with 5-fold excess (mol) of HyNic whichhad been dissolved in anhydrous DMF at 25 mg/mL. Following a 2.5 hourincubation at 24° C., excess linker was removed via desalting/bufferexchange over a 5 mL Zeba column into CB. A 2-SB A350 assay performed asdescribed above indicated an MSR of 3.4.

To the HyNic-dextran solution was added a stoichiometrically-limitingamount of 5′-4FB-oligonucleotide (0.5 mol-equivalents) to limit theaverage number of oligos per dextran to <1. Conjugation was allowed toproceed overnight at 4° C. before removal of free oligonucleotide bysize exclusion chromatography over an HR-10/30 Superdex 200 PG column.Mobile phase for the purification was Loading Buffer (20 mM HEPES, 25 mMsodium chloride, pH 7.0) at 1 mL/minute flow rate.

Unconjugated dextran was removed from the conjugated product usingVivapure Q Mini-H ion-exchange devices (Sartorius Stedim Biotech,France). Crude conjugate was loaded onto the devices in loading bufferand washed with 2×400 μL of the same to remove free dextran. Theoligonucleotide-dextran conjugates were eluted from the support withincreasing salt concentrations of 90 mM, 450 mM, and 750 mM sodiumchloride in loading buffer. Most of the conjugate eluted in the 450 mMand 750 mM fractions, and was pooled to afford the purified product. Theamino-dextran-oligo heterodimer was desalted and exchanged intomodification buffer using a 5 mL Zeba column in preparation for dyelabeling.

h. Dye-Labeling of Amino-Dextran-Oligo Heterodimers

To oligo-dextran-amino heterodimer at 6 mg/mL in Modification Buffer wasadded a 10.7-fold excess (mol) of DyLight dye NHS ester (Dyomics,Germany) with rapid mixing at pH 7.4. Dye labeling of the amino-dextranwas achieved over a 3 hour incubation at 24° C., at which time thereaction was placed into dialysis vs. several changes of PBS. Degree OfLabeling (DOL) was determined by dividing the concentration of dye bythe concentration of oligo (mol/mol), as determinedspectrophotometrically at A260 after correcting for the UV contributionof the dyes themselves.

i. Cell Preparation and Labeling

C57BL/6 mice were bred and housed in a specific pathogen-free facilitymaintained by the University of Chicago Animal Resources Center(Chicago, Ill.), and used under the guidelines of the InstitutionalAnimal Care and Use Committee (IACUC). Spleens were isolated andprocessed into single cell suspensions by pressing minced tissue througha fine mesh nylon filter, followed by washing with culture mediumconsisting of DMEM supplemented with HEPES, non-essential amino acids,penicillin/streptomycin and β-mercaptoethanol. Erythrocytes were lysedby brief incubation in a buffered ammonia chloride solution. Leukocyteswere suspended in DMEM supplemented with 5% fetal calf serum and brieflystored at 4° C. until being counted for the number of live cells.

To prepare cells for antibody staining, splenic leukocytes werealiquoted at a density of 0.5-1.0×10⁶ cells/sample in a bufferconsisting of 1×PBS with 1% BSA. Non-specific binding of IgG to cellularFc receptor was blocked by incubation in 50 μL of anti-FcR (clone 2.4G2hybridoma supernatant) for 20 minutes at 24° C.

Antibody-fluorophore labeled targeting hybrids were prepared in solutionprior to staining the cells by mixing antibody:oligo targetingconstructs (0.1-1 μg) with complementary oligo:fluorophore labelingconstructs in 1% BSA-PBS for 15-30 minutes at 24° C. Hybrids were thenadded to prepared, FcR-blocked murine splenocytes for 1 hour at 4° C.with slow rotation. Cells were washed once in 500 μL PBS to removeexcess hybrid, and analyzed by flow cytometry.

j. Preparation and Analysis of Quantitative Microspheres

4FB-modified 3 um paramagnetic particles (SoluLink Biosciences, SanDiego Calif.) were conjugated to HyNic-oligonucleotide at 0-20 nmol permg by 2 hour incubation in CB to result in oligo-surfaced microspheres(oligospheres). Oligospheres were washed in PBS to remove free oligo andstored in PBS at 4° C.

To prepare fluorophore-hybridized oligospheres, complementaryoligo:polyfluor was added to 6.25-50 μg microspheres (˜50-400×10³particles) in desired titrations (0-40 pmol/μg). Hybridization proceededin PBS for 15-30 minutes with gentle vortexing, spheres were washed toremove unbound fluorophore, and then were loaded into a black 96-wellplate for fluorimeter analysis (Tecan Safire 2, Switzerland).Oligosphere fluorescence was evaluated in the microplate vs a standardcurve of oligo:polyfluor labeling construct ranging from 0.0-1.0 pmolper microwell. Microspheres and labeling construct standards were eachdiluted in 100 μL total volume of dilution buffer (1×PBS) per microwell.Fluorimeter readings were normalized by using PBS dilution buffer as ablank for the standard curve, and unhybridized oligospheres to correctfor autofluorescence of the oligospheres. Standard curves for each offour DyLight fluorophores (Dy490, 549, 649, 405) were plotted usinggraphing software with X=fluorimeter intensity and Y=pmol labelingconstruct per sample. R² values were >0.98 for all four standard curves.

Following fluorimeter analysis, the number of microspheres per microwellwas enumerated using a handheld particle counter (Scepter CountingDevice; Millipore, Billerica, Mass.). Labeling construct per microwell(pmol) was converted to molecules labeling construct and divided by thenumber of particles per microwell to determine Label Per (microsphere)Event (LPE).

k. Flow Cytometric Instrumentation and Analysis

Most analyses were performed using a 4-laser, 12-detector BD LSRII flowcytometer (Becton Dickinson, San Jose Calif.) which is routinely alignedand calibrated for PMT linearity by the University of Chicago FlowCytometry Core Facility. Instrument layout includes one octagonal andthree trigonal optical arrays, each equipped with a single laser. Foranalysis of oligospheres for spectral compensation, a 3-laser,8-detector BD LSRII flow cytometer was used. For all analyses, cytometeracquisition settings were initiated prior to each experiment, and wereunchanged for the duration of analysis. Single-event data were acquiredusing FACSDiva software (BD, San Jose Calif.). Data were saved aslist-mode data files (.fcs) and analyzed using FlowJo software(TreeStar, Ashland, Oreg.).

Each leukocyte sample, consisting of a minimum of 10,000 events, wasscatter-gated on the lymphocyte population according to standard methods[24] using an unstained control sample prior to interpretation ofresults. Microspheres were also scatter-gated to define single events(doublet exclusion).

l. Pseudocode for Quantitative Flow Software Algorithms

Main Program: Ask User to input the Number of Channels: Store valueNumber_of_Channels Ask User to upload Control_LPE Lot and IntensityNumbers (*.csv): Store in Control_LPE CALL subroutine Zero_LPE(Number_of_Channels) return Zero_GMFI.Channel Array CALL subroutineStandard_Curve (Number_of_Channels, Zero_GMFI.Channel Array) returnStandard_Curve.Channel Array FOR Channel equals 1 to Number_of_ChannelsDO Set Exp_ABC.Channel equal to 0 // Antibody Binding per Cell SetExp_GMFI.Channel equal to 0 Set Exp_LPE.Channel equal to 0 Set Zero_Flagequal to FALSE REPEAT Ask User to Gate Experiment Cells Load GeometricMean Fluorescence Intensity (GMFI) IF GMFI is between 10² and 10⁵ DO SetExp_GMFI.Channel equal to GMFI Set Zero_Flag equal to TRUE UNTILZero_Flag equals True Plot Standard_Curve.Channel Set Exp_LPE.Channelequal to y=mx^(n) where X equals Exp_GMFI.Channel Set Exp_ABC.Channelequal to Exp_LPE.Channel Output Standard_Curve.Channel Plot andExp_ABC.Channel to User Zero_FPE subroutine: FOR Channel equals 1 toNumber_of_Channels DO Set Zero_GMFI.Channel equal to 0 Set Zero_Flagequal to FALSE REPEAT Ask User to Gate Unstained Cells (Zero reading)Load Median Fluorescence Intensity (GMFI) IF GMFI is between 0 and 10²DO Set Zero_GMFI.Channel equal to GMFI Set Zero_Flag equal to TRUE UNTILZero_Flag equals True Standard_Curve subroutine: FOR Channel equals 1 toNumber_of_Channels DO Set Cell_GMFI.Channel equal to 0 Set Zero_Flagequal to FALSE REPEAT Ask User to Gate Control Cells Load Geometric MeanFluorescence Intensity (GMFI) IF GMFI is between 10² and 10⁵ DO SetControl_GMFI.Channel equal to GMFI Set Zero_Flag equal to TRUE UNTILZero_Flag equals True Ask User if they want to Compensate? (Yes/No) IFYes DO Have FlowJo software Discard Peak Option Plot GMFI versus LPE SetStandard_Curve.Channel equal to y=mx^(n) where X equalsCell_GMFI.Channel

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of quantitative flow cytometry comprising: (a) contacting asample comprising one or more cells with a labeled targeting constructunder conditions suitable for binding of the labeled targeting constructto an antigen on the cells; wherein the labeled targeting constructcomprises a targeting moiety:ligand complex and a bioconjugate; whereinthe bioconjugate comprises a biomolecule attached to a labeling moiety;and wherein the biomolecule of the bioconjugate binds to the ligand ofthe targeting moiety:ligand complex; (b) analyzing the cells bound tothe labeled targeting construct in the sample in a flow cytometer; (c)analyzing a population of quantitative labeled ligand-surfacedmicrospheres, wherein the population of quantitative labeledligand-surfaced microspheres is labeled with the same bioconjugate asthe labeled targeting construct, in the flow cytometer; and (d)quantifying the amount of labeled targeting construct binding per cell;and wherein the ligands of the ligand-surfaced microsphere and theligand of the targeting moiety:ligand complex are peptides or haptens.2-31. (canceled)
 32. A method of quantitative flow cytometry comprising:(a) contacting a sample comprising one or more cells with a labeledtargeting construct under conditions suitable for binding of the labeledtargeting construct to an antigen on the cells; wherein the labeledtargeting construct comprises a targeting moiety:ligand complex and abioconjugate; wherein the bioconjugate comprises a biomolecule attachedto a labeling moiety; and wherein the biomolecule of the bioconjugatebinds to the ligand of the targeting moiety:ligand complex; (b)contacting the sample with a population of quantitative labeledligand-surfaced microspheres, wherein the population of quantitativelabeled ligand-surfaced microspheres is labeled with the samebioconjugate as the labeled targeting construct; (c) analyzing thepopulation of quantitative labeled ligand-surfaced microspheres and thecells that bind the labeled targeting construct in the sample using aflow cytometer; (d) determining a GMFI versus LPE trendline from theGMFIs of the population of quantitative labeled ligand-surfacedmicrospheres; (e) determining the LPE for one or more cell populationsthat bind the labeled targeting construct from the GMFI versus LPEtrendline; and (f) quantifying the amount of labeled targeting constructbinding per cell, wherein the biomolecule of the bioconjugate binds tothe ligand of the ligand-surfaced microsphere; wherein the target is acellular target on or within cells; and wherein the ligands of theligand-surfaced microsphere and targeting moiety:ligand complex arepeptides or haptens.
 33. The method of claim 32 further comprising: (a)contacting the sample with at least a first and a second labeledtargeting construct, wherein the first labeled targeting constructcomprises a targeting moiety:ligand complex and a bioconjugate thatdiffers from the targeting moiety:ligand complex and bioconjugate of thesecond labeled targeting construct, under conditions suitable forbinding of the first and the second labeled targeting constructs totheir respective targets on or in the cells; and (b) contacting thesample with at least a first and a second population of quantitativelabeled ligand-surfaced microspheres, wherein the bioconjugates of thefirst and the second populations of quantitative labeled ligand-surfacedmicrospheres differ from each other, but are the same as thebioconjugates utilized in the labeled targeting construct of either thefirst or the second labeled targeting constructs. (c) analyzing thepopulations of quantitative labeled ligand-surfaced microspheres and thecells that bind the labeled targeting construct in the sample using aflow cytometer; (d) determining the GMFI versus LPE trendline from theGMFIs of at least two different populations of quantitative labeledligand-surfaced microspheres; (e) determining the LPE for the one ormore cell populations that bind the first and/or second labeledtargeting construct from the GMFI versus LPE trendlines; and (f)quantifying the amount of labeled targeting construct binding per cell.34-75. (canceled)
 76. An interchangeable labeling system comprising: (a)an antibody:ligand targeting complex comprising an antibody region and aligand region; (b) a plurality of different bioconjugates comprising alabeling moiety and a biomolecule region that binds to the ligand regionof the targeting moiety:ligand complex, wherein each of the plurality ofdifferent bioconjugates has a different labeling moiety, but comprisesthe same biomolecule region; wherein the ligand region comprises apeptide or hapten. 77-121. (canceled)
 122. The method of claim 33,wherein the sample is a whole blood sample or a buffy coat sample. 123.The method of claim 32, wherein the sample is a cultured preparation ofmammalian cells, a biopsy cell aspirate, a tissue sample, or anenvironmental sample.
 124. The method of claim 32, wherein the cells areimmune cells.
 125. The method of claim 124, wherein the immune cells areT cells, B cells, NK cells, granulocytes, or monocytes.
 126. The methodof claim 32, wherein the cells are tumor cells, stem cells orimmortalized cells.
 127. The method of claim 32, wherein the cells arerodent, plant, bacterial, fungi, protozoan, or metazoan cells.
 128. Themethod of claim 32, wherein the labeled targeting construct comprises anantibody that specifically binds to CD4, CD8, CD43, or CD62L.
 129. Themethod of claim 33, wherein the first labeled targeting constructcomprises an antibody that binds to CD4 and the second labeled targetingconstruct comprises an antibody that binds to CD8.
 130. The method ofclaim 33, further comprising contacting the sample with at least a thirdand a fourth different labeled targeting construct under conditionssuitable for binding of the third and the fourth labeled targetingconstructs to their respective targets on the cells.
 131. The method ofclaim 130, wherein the first labeled targeting construct comprises anantibody that binds to CD4, the second labeled targeting constructcomprises an antibody that binds to CD8, the third labeled targetingconstruct comprises an antibody that binds to CD43, and the fourthlabeled targeting construct comprises an antibody that binds to CD62L.132. The method of claim 32, wherein the targeting moiety is an antibodyand wherein the antibody is a monoclonal antibody, an antibody fragment,a polyclonal antibody, a recombinant antibody, a synthetic antibody, ora chimeric antibody.
 133. The method of claim 32, wherein the labelingmoiety comprises a fluorescent label and wherein the fluorescent labelis Dy490, Dy549, Dy649, or Dy405.
 134. The method of claim 32, whereinthe population of quantitative labeled ligand-surfaced microspherescomprises at least four subpopulations of different ligand-surfacedmicrospheres bound with at least four different concentrations ofbioconjugates.
 135. The method of claim 134, wherein the labeling moietycomprises a fluorescent label and wherein the fluorescent label isDy490, Dy549, Dy649, or Dy405.
 136. The method of claim 32, wherein thelabeled targeting construct is contacted with the sample before or afterthe population of quantitative labeled ligand-surfaced microspheres iscontacted with the sample.
 137. The method of claim 32, wherein thebiomolecule is an antibody.